BACKGROUND
h/aca ribonucleoprotein particles  in eukaryotes consist of four highly conserved core proteins and one molecule of h/aca small nucleolar rna , and most of them direct pseudouridylation of target rnas at specific sites .
in this process, one of the core proteins acts as a pseudouridine synthase, while the h/aca snorna selects the residues to be isomerized via specific base-pairing.
proteins catalyzing the conversion of uridines to pseudouridines belong to a highly conserved family, well-characterized examples of which include archaea, yeast and trypanosome cfb5p  <cit> , drosophila mfl/nop60b  <cit> , rat nap <dig>  <cit> , and mouse and human dyskerin  <cit> .
in eukaryotes, these proteins accumulate in the nucleolus and participate in various cellular functions including processing and modification of ribosomal rna  and maintenance of telomere integrity in mammals .
genetic depletion experiments in different organisms have invariably shown that these proteins are essential for viability  <cit> , indicating that they have important biological roles.
the finding that human dyskerin is involved in two congenital diseases further supports this notion.
mutations in these proteins are responsible for x-linked dyskeratosis congenita   <cit>  and for hoyeraal-hreidarsson syndrome, now recognized as a severe dc allelic variant  <cit> .
functional conservation of pseudouridine synthases is so remarkable that the archaeal acbf5p protein has recently been shown to assemble efficiently with a yeast h/aca snornp core component, nop <dig>  and with human telomerase rna, which has a h/aca box motif  <cit> .
the biological role of rna pseudouridylation is still debated.
it has been suggested that it contributes to rrna folding, rrnp assembly and ribosomal subunit assembly.
subtle enhancing of ribosomal functions such as codon recognition has also been proposed  <cit> .
remarkably, recent data indicate that mutations in mammalian dyskerin impair translation from ires  elements, thus specifically affecting cap-independent translation of a subset of mrnas  <cit> .
however, the role of h/aca snornps extends beyond ribosome biogenesis.
in fact, although rrna is the most common modification target, spliceosomal snrnas or trnas can also be modified  <cit> .
furthermore, "orphan" snornas that lack complementarity with rrna or snrnas have also been described, and it is plausible that they target cellular rnas that remain unidentified.
since proteins of the cbf5p family are essential for biogenesis and accumulation of h/aca snornas, mutations in them might trigger diverse effects.
for example, different mutations of human dyskerin have been associated with reduced levels of distinct subsets of h/aca snornas  <cit> , raising the possibility that some pathological aspects of dc may be related to the particular functions of the specifically-affected forms.
indeed, the repertoire of functions attributed to members of the cbf <dig> family is wide and continually increasing; these genes may be involved in many biological processes.
in yeast, cbf5p was first described as a low-affinity centromeric dna binding protein  <cit> .
subsequently, depletion of cbf5p was shown to cause nucleolar fragmentation and to disrupt the nucleolar localisation of trna  <cit> .
in mice, dkc <dig> alleles carrying hypomorphic mutations led with very high frequency to tumour development, indicating that the gene acts as a potent oncosuppressor  <cit> .
a novel role in snorna metabolism has recently been reported for the yeast and mammalian proteins: they are directly involved in the early steps of h/aca snornp assembly, since they are cotranscriptionally recruited at the 3' end of the nascent h/aca snornas  <cit> .
in trypanosomes, rnai-induced silencing of cbf <dig> has been shown not only to eliminate pseudouridylation on the spliced leader rna , but also to abolish its modification at the fourth cap- <dig> nucleotide  <cit> .
as a result of defects in the sl rna and decreased modification of the u small nuclear rnas, trans-splicing was inhibited at the first step of the reaction.
considering the wide range of biological effects directed by members of the cbf <dig> gene family, it is plausible that this functional complexity might rely, at least in part, on the production of multiple transcripts with different properties.
since a complex expression pattern is often observed for multifunctional genes in higher eukaryotes, we planned to analyse the molecular organization and transcriptional activity of the drosophila orthologue in greater detail, with the aim of better defining its coding properties and shedding new light on its complex functions.
in previous studies, we isolated the gene encoding drosophila pseoudouridine synthase, called minifly   <cit> , and showed that the use of alternative 3' utrs resulted in two main mrnas,  <dig>  and  <dig>  kb in length.
these two species had different expression profiles, with the  <dig>  kb mrna constitutively expressed in both sexes throughout the life cycle, and the  <dig>  kb species mainly expressed in females and maternally transmitted to the developing embryos  <cit> .
however, the two transcripts had identical protein coding potentials and differed only in their 3' untranslated regions .
as with genes encoding proteins involved in the synthesis, structure or function of the translational apparatus, mfl was shown to belong to the 5' top family   <cit> .
members of this family share a c residue at the + <dig> position followed by a 5– <dig> nt polypyrimidine tract in a short 5' non-translated region; their mrnas typically show growth-dependent translational regulation  <cit> .
we also showed that mfl hosts a snorna gene of the h/aca class within one of its introns.
this gene, named snoh <dig>  directs pseudouridylation of drosophila 18s rrna at position u <dig>  <cit> , modification of which residue is conserved from yeast to man.
in this paper we report that snoh <dig> is just one member of a variegated cluster of small ncrna genes hosted within mfl introns, revealing that this gene exhibits a much more complex overlapping coding/noncoding architecture than previously suspected.
in addition, we show that mfl produces two novel coding transcripts.
while one of these mrnas is characterized only by an extended 3'utr, the second is alternatively spliced, accumulates mainly in females, and encodes a rare variant protein that is predicted to have distinct functional properties.
so far, mfl is the only eukaryotic pseudouridine synthase gene for which multiple mrnas have been reported, and the description of the variant protein and the various ncrnas it can encode may provide useful insights into the various functions of other members of this conserved family.
RESULTS
identification of two novel mfl coding transcripts
in previous primer-extension experiments we detected and mapped a single mfl transcription start site  <cit>  .
we also noticed that a very short upstream region of about  <dig> bp separates mfl from the close, divergently-transcribed gene mrp <dig>  on the basis of these observations, we considered that further transcript heterogeneity is more likely to derive from the alternative 3' ends.
therefore, the transcriptional activity of the gene was first re-examined by 3'-race .
based on the structures of the previously-described  <dig>  and  <dig>  kb mrnas  , internal primers were derived from the sequence of exons  <dig>   <dig>   <dig> and  <dig>  and used to search for additional poly sites, using poly+ rna extracted from male and female adult flies as template.
in addition to the two expected bands, a longer product was obtained in each 3'-race reaction, revealing the presence of a poly site further downstream .
these additional products were markedly more abundant in the female rna preparation, implying a sex-bias in the use of this novel 3' site.
subsequent nucleotide analyses of these amplification products confirmed that mfl encodes a third mrna that differs from the  <dig>  and  <dig>  kb species only in the presence of three additional exons in a longer 3'utr  .
northern analysis of the poly+ rna preparations probed with the novel downstream exons revealed a transcript of about  <dig>  kb, expressed mainly in females .
this is the expected length for an mrna species starting at the previously-mapped 5' site and terminating at the novel 3' end, further confirming that the  <dig> ,  <dig>  and  <dig>  kb mrnas are distinguished solely by 3'-end heterogeneity.
we then searched for cis-acting control elements affecting mrna stability, location or translation within these overlapping 3'utr sequences.
however, no functional motif currently annotated in the utresource database  <cit>  was identified in any of these regions.
further experiments will therefore be required to establish whether these sequences include still-unidentified regulatory motifs.
strikingly, northern analyses of poly+ rna revealed that a large polyadenylated transcript of about  <dig>  kb also derives from this downstream region .
this species had already been detected in adult female or embryonic rna preparations by more upstream mfl genomic probes  <cit> , but previous attempts to isolate cdna clones representing this form proved unsuccessful even after extensive screening of various cdna libraries.
since in the present set of experiments we again failed to identify any product representing this rna, its structure remains elusive, so we cannot exclude the possibility that this species represents a variant transcript extending much further, or an unprocessed rna precursor.
we next tried to connect the novel 3' exon cluster with more upstream exons by rt-pcr, with the aim of detecting alternative splicing events within the coding sequence of the gene.
for this purpose, we used poly+ rna from male and female adult flies as template, and various combinations of forward primers positioned inside the coding region  with reverse primers positioned along the  <dig>  kb-specific 3'utr exons .
in each combination, primers derived from exons  <dig> or  <dig> always yielded a single amplification product, markedly more abundant in females; sequence analysis invariantly confirmed the structure of the  <dig>  kb mrna previously obtained by 3'-race.
in contrast, two types of products were always obtained when a forward primer from exon  <dig>  in each combination of reverse primers, was used to reverse-amplify female rna.
in addition to the expected band, a minor product of smaller size was noticed in each reaction.
as an example, the result of an exon 2– <dig> rt-pcr amplification from female rna is reported in fig.
2a.
in this case, in addition to the  <dig> kb product expected from the  <dig>  kb mrna, an additional product of  <dig> bp was observed.
sequence analysis of the additional shorter products obtained in this set of experiments revealed an alternative splicing event that joins the 5' splice donor of exon  <dig> to the 3' splice acceptor of exon  <dig>  to confirm the presence of this spliced mrna, the same 5' primer was used in combination with either a 3' primer spanning the 3– <dig> exon junction, able to detect only the alternatively spliced rna subform, or one spanning the 5– <dig> exon junction, able to detect the whole set of transcripts generated by the canonical splicing pattern comprising the  <dig>  kb constitutive mrna and the  <dig>  and  <dig>  kb maternal species.
both primer pairs yielded positive amplification of a fragment with the expected size; moreover, both products were obtained at a significantly higher level in females, indicating that, as previously described for the  <dig>  kb  <cit>  and also for the  <dig>  kb species described above, the variant mrna exhibits a sex-preferential expression profile .
a full-length cdna clone representing this novel mrna was obtained by combining 5' and 3' race experiments.
gene-specific primers were designed to amplify the 5' end of the transcript and to give fragments partially overlapping with the 3'-race products .
a short overlapping sequence in the 5' and 3' products allowed us to construct a virtually full length cdna by restriction digestion at the common bamh1site.
the fragment obtained was about  <dig> kb and its nucleotide sequence  indicated that the novel mrna starts at the unique, previously-mapped transcription start site  <cit> .
intriguingly, this transcript is characterized by the skipping of five internal exons and the absence of any internal stop codon, indicating that it may encode a novel protein subform .
given that the  <dig>  kb mrna species was barely detected in the northern analyses of poly+ rna preparations, we used quantitative real-time pcr to check its relative abundance in the poly+ rna preparations obtained from various sources: adult females, manually dissected adult ovaries and cultured schneider  <dig>  drosophila cells.
the relative abundances of the variant and canonically-spliced mrnas  were compared among the three samples after normalization against αtub84b expression.
the  <dig>  kb mrna was significantly more abundant in ovaries than in whole adult females and its accumulation was at least nine-fold greater; the canonically-spliced mrna was also slightly enriched in this organ.
in proliferating s <dig> cells, the alternatively-spliced species was markedly more abundant, whereas the level of the canonically-spliced mrnas was slightly reduced .
structural properties and expression of the mflα novel protein subform
the rare alternatively-spliced mfl mrna is predicted to encode a variant protein of  <dig> amino acids, with a molecular mass of  <dig>  kda, which we named mflα.
the amino acid sequence of this variant protein is shown in fig.
4a, aligned below that of mfl, identically encoded by the  <dig> ,  <dig>  and  <dig>  mrnas, and that of human dyskerin.
mflα fully overlaps the mfl sequence at its amino-terminal region, where both proteins exhibit an identical tract of  <dig> aa that includes the n-terminal nuclear location signal , and the two highly-conserved trubi and trubii motifs that share homology with bacterial and yeast trna pseudouridine synthases and are directly involved in the pseudouridylation process.
however, mflα exhibits a unique c-terminal tract of  <dig> residues that shows no significant homology with any known functional motif.
instead, this region replaces a large carboxy-terminal portion of the mfl protein that includes at least three domains for which a functional role has been proposed.
the first missing motif corresponds to the pua domain, an rna binding motif observed in several families of archaeal, bacterial and eukaryotic rna-modifying proteins including pseudouridine synthases and rrna methylases.
this domain has also been found in bacterial and yeast glutamate kinases, as well as in families of eukaryotic proteins that are thought to act as translation factors  <cit> .
intriguingly, it has recently been shown to play a crucial role in archaeal snornp assembly, and to be necessary for acbf5p binding to guide rnas  <cit> .
the second missing domain is represented by a block of more than twenty residues with a central tyrosine  that is identical in mfl and human dyskerin and is highly related to the uracil-binding pocket in uracil-dna glycosylases  <cit> .
finally, mflα also lacks a highly-charged lysine-rich carboxy-terminal region that contains an overlapping bipartite nls, raising the possibility that this variant protein may have a different subcellular distribution, or that it is less tightly retained in the nuclei.
collectively, these distinctive features suggest that mflα may have a distinct role in at least some of the essential cellular activities of mfl.
as a preliminary step to investigating this possibility, we attempted to assess the effective in vivo accumulation of this rare subform.
on the basis of the observations indicating a higher abundance of the  <dig>  kb mrna in s <dig> cells, we selected this source for checking the presence of mflα protein by western blotting.
rabbit polyclonal antibodies were raised against two peptides, both included in the common n-terminal region of mfl/mflα , and protein extracts from s <dig> cells were subjected to western analysis with these antibodies.
in addition to the canonical mfl protein, a less abundant band of the molecular weight expected for mflα was detected , indicating that the alternatively-spliced mrna may encode a variant protein.
a cluster of small ncrna genes is intron-encoded at the mfl variable 3' utrs
to check mfl transcription further, we performed northern blotting of total rna preparations using genomic probes spanning the variable mfl 3' ends.
the results showed that a set of small rna molecules, about  <dig> nt long and highly abundant in females, also derive from this region.
to map these molecules in greater detail, shorter probes having either exonic or intronic localisation were used to analyse rna preparations from adult flies of both sexes .
probes derived from introns  <dig>   <dig>   <dig> and  <dig> all detected the  <dig>  kb species .
they also detected a radioactive signal of about  <dig> nt, as estimated by carefully assessing the rna length on 6% denaturing polyacrylamide gels and by mapping the 5' end by primer extension analysis .
inspection of the mfl 3' genomic sequence  indicated that introns  <dig>   <dig>   <dig> and  <dig> each host a copy of dmsnr <dig> , a snorna gene of the c/d family previously identified in the course of a genome-wide computational search  <cit> .
the strong cross-hybridization with rrna shown by these intronic probes  is essentially explained by their perfect complementarity to the 28s rrna molecule, which is recognized by long d and d' antisense elements .
as shown in the figure, probes spanning introns  <dig> and  <dig> detected rna molecules estimated at  <dig> nt, again on the basis of 6% denaturing polyacrylamide gel electrophoresis and primer extension analysis of the 5' end, in addition to the  <dig>  and  <dig>  kb mrnas.
nucleotide analysis of introns  <dig> and  <dig> revealed that each hosts a copy of a small ncrna gene, the sequence of which substantially overlaps that of the dmorc/d_ <dig> a-b molecules  previously described by huang et al.
<cit> .
these molecules exhibit canonical d and d' boxes  but have a degenerate c motif with a sequence varying between the two tandemly-repeated isoforms.
the biological functions, if any, of these molecules remained to be firmly established, so they were classified as orphan c/d snornas  <cit> .
indeed, we noticed that they lack a nucleolar- or cajal body- specific location , so in accordance with widely-accepted nomenclature  <cit> , we refer to them as small non-messenger rnas derived from polytene region  <dig> .
the two copies of snm <dig>   share 85% overall sequence conservation and exhibit a  <dig> bp internal segment of perfect identity .
as judged by southern blotting  and a computational search on the genome sequence, no other snm <dig> copy is present elsewhere in drosophila.
organization of the ncrna gene cluster mapped at the mfl 3'-utrs conformed to the one-gene-per-intron rule widely observed in animal genomes.
moreover, we noticed that the tandemly-clustered dmsnr <dig> genes are all located about  <dig> nt upstream the 3' splice site of their host introns.
in vertebrates, a position about  <dig> nt upstream the 3' splice site is reportedly optimal for expression of intronic c/d box snornas, since this distance may furnish optimal synergy with splicing, favouring c/d box snornp assembly  <cit> .
the conserved position of dmsnr <dig> isoforms indicates that the rule observed by hirose and coworkers  <cit>  may also be operative in invertebrates.
dmsnr <dig> genes, first described by our group  <cit> , were reported in a subsequent genome-wide analysis to be generated, along with dmorc/d_ <dig> a-b molecules, by introns of a long, polyadenylated non-coding host transcript named duhg  <dig>  <cit> .
however, identification of the additional mfl poly+ site in the present study provides clear evidence that dmsnr <dig> and dmorc/d_9/snm <dig> copies are all intron-encoded by this protein-coding gene.
an intriguing functional consequence of the mfl coding/non-coding arrangement is that pre-mrnas ending at different alternative poly  sites can release distinct sets of nested ncrnas.
in fact, a pre-mrna molecule ending at the first poly site can exclusively produce the h/aca snorna h <dig>  while one ending at the middle site may also release three dmsnr <dig> isoforms .
a pre-mrna terminating at the most downstream site may instead produce three different types of small ncrnas, including snoh <dig> and all the dmsnr <dig>  and snm <dig> isoforms.
strikingly, the ability to release snm <dig> molecules is restricted to mfl transcripts ending at the last poly  site, so that production of these ncrnas is predicted to be coupled with that of the  <dig>  and  <dig>  kb mrnas.
in conclusion, alternative 3'-ends allow mfl to produce not only two distinct protein subforms, but also different ncrnas that may potentially contribute to its biological functions.
expression and function of mfl intron-encoded ncrnas
all the dmsnr <dig> isoforms  possess canonical c , d and d'  boxes, suggesting that they encode bifunctional snornas of the c/d family .
long tracts of perfect complementarity to drosophila 28s rrna were found upstream of both the d and d' boxes  <cit> .
since c/d snornas invariably select the nucleotide positioned  <dig> base pairs upstream of the d/d' box for methylation  <cit> , these antisense elements were predicted to modify, respectively, the g <dig> and am <dig> residues , two methylation sites conserved between yeast and vertebrates.
in fact, methylation at the g1083-equivalent residue is known to be guided by yeast snr <dig> and mammalian u <dig> snornas, while am <dig> modification is directed by yeast snr <dig> and by a still-unidentified mammalian snorna  <cit> .
methylated residues have not yet been experimentally mapped on drosophila rrna, so we attempted to detect effective modification at the predicted sites by reverse transcription of the specific rrna sequence at low dntp concentration, as described by maden  <cit> .
when this method is used, a specific reverse transcription stop occurs on  a 2'-o-methylated nucleotide at low, but not at elevated, dntp concentrations.
as shown in fig 6b, this experiment confirmed the presence of methylated nucleotides at both predicted positions.
strikingly, one additional 2'-o-methylation was detected, at position gm <dig> of 28s rrna .
modification at this site has not yet been described in other organisms and may be specific to drosophila rrna.
since no other drosophila snorna that may be able to methylate the g <dig> and the am <dig> residues specifically has so far been described, modification of these sites strongly supports the functional role of the dmsnr <dig> molecules as rrna methylation guides.
in contrast, the function of the snm <dig> molecules is hard to guess, and further experiments are required.
to characterize further the small ncrnas originating from the mfl 3' region, a panel of total rna samples extracted from various stages of drosophila development or from the s <dig> cell line was subjected to northern blot analysis with probes specific for snoh <dig>  dmsnr <dig> or snm <dig>  these intron-encoded ncrna genes were all actively expressed in s <dig> cells, and throughout the drosophila life cycle they exhibited a very similar expression pattern .
as shown in fig.
<dig>  the ncrnas accumulate constitutively throughout development, but their expression is quantitatively modulated, reaching the highest levels in young larvae and in adult females.
it should be noted that the marked sex-bias in snoh <dig> and dmsnr <dig> expression is quite unusual for snornas.
considering that these snornas are devoted to modifying, respectively, the 18s and 28s rrnas, it is plausible that the higher levels in females may essentially be due to the higher level of protein synthesis in the ovaries, which in adult females may constitute more than half the body weight.
to address the function of dmsnr <dig> and snm <dig> further, we next investigated their intracellular distribution by in situ hybridization.
given that these rnas accumulated at higher levels in larvae and in adult females, we selected the intestine as a larval organ and the ovary as an adult organ for these analyses.
when specific digoxigenin-labelled antisense probes were used to analyse whole-mount intestine preparations, the dmsnr <dig> molecules were shown to be expressed in all cells .
within ovarioles, these molecules were also expressed ubiquitously, being detected at all stages of oogenesis in both the nurse and the follicle cells .
moreover, dmsnr <dig> molecules accumulate specifically in the nucleoli, as substantiated by observation of the large nuclei of the polyploid nurse cells.
these cells develop unusual nucleoli comprising a shell of interconnected fibres around the nuclear periphery  <cit> .
as is evident from the figure, the dmsnr <dig> hybridization signal in the nurse cells is specifically concentrated in these nuclear peripheral structures, as expected for bona fide snornas .
in contrast, snm <dig> expression in the ovarioles appeared to be restricted to late oogenesis, starting from stage  <dig> ; moreover, these rnas accumulated specifically in the nurse cells, not the follicle cells.
within the nurse cells, the snm <dig> molecules appear to be concentrated in the nucleoplasm but are not located within the nucleoli.
these ncrnas also show an intriguing expression pattern in the larval intestine, where in most microscopic fields they are detected only in subsets of cells that appear to be actively dividing .
most cells expressing snm <dig> rnas are in fact yet-unseparated daughter cells just exiting from mitosis, or paired cells that plausibly derive from the same mitotic event, raising the possibility that expression of these molecules might be regulated during the cell cycle.
an obvious conclusion from these in situ experiments is that spatial and temporal expression of dmsnr <dig> and smn <dig> genes are differentially regulated.
it is possible that smn <dig> molecules may be independently transcribed from a specific alternative promoter, or that their biogenesis and/or stability may depend on the presence of specific factors.
whatever the case, such fine regulation was unexpected and makes it unlikely that these small rnas merely represent transcriptional noise.
to investigate this aspect further, we checked the phylogenetic conservation of smn <dig> genes by searching for their expression in drosophila-related species such as d. yakuba, d. virilis and d. ananassae.
by northern blot experiments, we were able to detect these ncrnas only in the most closely-related species d. yakuba , which belongs to the melanogaster subgroup.
in contrast, the dmsnr <dig> molecules were positively detected in all three species.
a comprehensive picture of sequence conservation of the mfl 3' genomic region is presented in fig.
<dig>  which shows a diagram of multiple genome alignments obtained by the ucsc genome browser  <cit> .
peaks in the conservation plot can be observed for each of the 3'utr introns, but strong conservation among all the annotated genomes of the drosophila species occurs only for dmsnr <dig> isoforms.
the weaker phylogenetic conservation displayed by snm <dig> suggests that these genes may have evolved more recently.
however, evolutionary conservation may not be a reliable signature for functional ncrnas, since many regulatory ncrna evolve quickly and may co-evolve with their functional targets  <cit> .
discussion
production of multiple transcripts with different coding/non-coding properties is known to contribute largely to the complexity of eukaryotic transcriptomes, and analysis of the full range of different transcripts that a gene can encode may provide important insight into its biological functions.
although alternative mrna subforms have never been described for any member of the cbf5/mfl/dkc <dig> family, transcripts that have very low abundance, or are expressed in selected cell types or in response to specific stimuli, may have escaped analysis.
indeed, rare products have important roles in many physiological processes and can often trigger crucial responses to developmental or growth stimuli.
the detailed analyses of the mfl gene reported here show that its molecular organization is much more complex than previously suspected and its coding potential is correspondingly expanded.
indeed, mfl is the only pseudouridine synthase gene for which extensive evidence of multiple transcripts has been reported so far.
our results showed that the canonical mfl protein can be encoded by three mrnas distinguished solely by 3' end heterogeneity, two of them displaying a maternal pattern.
these overlapping 3'-utrs may plausibly play a role in imposing different expression profiles on the mfl mrnas.
in this regard, an intriguing recent suggestion is that translation of 5'-top mrnas may become less stringent with increasing 3'-utr length  <cit> .
consistent with this notion, it could be surmised that the diverse lengths of 3'-utrs in the  <dig> ,  <dig>  and  <dig>  kb mrnas may per se influence the efficiency of their translation under different growth conditions; moreover, the  <dig>  kb mrna is the species for which the most stringent translational regulation should be expected.
the data reported in this paper also reveal that mfl encodes a novel alternatively-spliced protein subform, which represents the first example of a variant protein encoded by any member of the cbf <dig> gene family.
the main distinctive trait of this protein, named mflα, is its unique c-terminus, where a short tract of  <dig> amino acids replaces the large carboxy-terminal moiety of the major canonical protein, which includes the overlapping pua and tyr domains and the bipartite nls.
given that the pua domain of archaeal acbf5p has recently been shown to be essential for binding the guide rnas  <cit> , its lack is predicted to affect this functionally relevant feature, raising the possibility that mflα may participate in the formation of less efficient, or even inactive, h/aca snornps.
if this were the case, expression of this subform might have an autoregulatory role in mfl expression.
however, we cannot presently exclude the possibility that its specific c-terminus may allow mflα to interact with different efficiency or different specificity with distinct protein partners, eventually participating in the assembly of specific snornp subtypes.
on the other hand, neither can we exclude the possibility that this subform has so far unknown functions, even unrelated to those of the major canonical protein.
indeed, a further relevant distinctive trait of mflα is the absence of the c-terminal nls.
this might plausibly allow a subtly modified modulation of the rate of nuclear transport, or of nuclear retention, or of the subnuclear distribution of the mflα subform in response to a various cellular stimuli.
in this context, it is intriguing to note that deletion of the c-terminal lysine-rich cluster did not affect the nucleolar location of human dyskerin, but influenced the rate of transport into the nucleus  <cit> , while truncation of the c-terminal basic domain of yeast cbf5p led to delay at the g2/m phase of the cell cycle  <cit> .
finally, we noticed that the mfl c-terminal moiety deleted in mflα is particularly rich in putative phosphorylation sites.
since many cellular processes are controlled in a phosphorylation-and cell cycle-dependent manner, including protein synthesis and cell division, it is conceivable that mfl and mflα may respond in different ways to various growth conditions and cellular signals.
an unexpected and distinctive peculiarity of mflα concerns its greater accumulation in adult females.
although this sex-biased expression suggests that the subform is required during oogenesis and in the early stages of drosophila development, it would be premature to exclude the possibility that its expression may be identical in both sexes at different developmental stages, or that a sharply restricted or a transient expression profile may have hindered its detection in adult males.
indeed, the data reported in this paper show that higher levels of expression in females are a general feature of mfl transcripts, either coding or non-coding, the only exception being the  <dig>  kb constitutive mrna, which has the same abundance in both sexes  <cit> .
this observation adds further strength to a recent report that included mfl/nop60b among the list of drosophila genes that are significantly more strongly expressed in purified female germline stem cells  <cit> .
it is also worth noting that the levels of all mfl transcripts peak at the major sites of cell growth and division during the drosophila life cycle – late embryos, larvae and adult ovaries – making it plausible that gene expression might be linked to the rate of cell growth and proliferation.
this hypothesis is compatible with the essential role in ribosome biogenesis  <cit>  and with recent microarray analyses that indicated mfl/nop60b to be a major target of d-myc induction  <cit> .
in this light, the different levels of mflα expression between male and female adult flies may only be coincidental, simply reflecting the different rates of protein synthesis in adult flies of different sexes.
although further experiments are required to elucidate the molecular mechanisms that regulate mfl expression, the intriguing pattern displayed by snm <dig> molecules  in proliferating intestine cells is also in good agreement with this view.
the complex molecular organization described here for mfl may be of general relevance, possibly unravelling aspects common to other orthologues.
although no variant dkc <dig> transcript has been characterized thus far in mammals, it is intriguing that deletion and splice site mutations in the last exon of the gene have been found in dc patients  <cit> , and that a small deletion covering only the last dkc <dig> exon proved to be lethal in mouse knock-out experiments  <cit> .
together, these observations indicate that the 3' portion of the gene is also highly biologically relevant in mammals.
moreover, dkc <dig> -deleted alleles triggered early lethality only when they occurred on the maternal allele, revealing the existence of a maternal effect in the transmission of dc disease  <cit> .
it is tempting to speculate that, similarly to mfl, mammalian dkc <dig> genes may encode still-undetected transcripts, the expression of which may account for this sex-specific effect.
our data also show that  mfl introns harbour a variegated cluster of small ncrna genes comprising an h/aca snorna gene, four copies of the c/d snorna dmsnr <dig> and two copies of the small ncrna of unknown function.
the structure of this dense cluster conforms to the one ncrna gene per intron rule widely observed in animal genomes, and strongly implies that several duplication events have occurred during its evolution.
this coding/non-coding genetic arrangement has obvious regulatory potential: to coordinate the expression of nested ncrnas with the protein products of the gene.
common functional roles are often shared by host genes and intron-encoded ncrnas.
in the case of mfl, the snorna h <dig> and dmsnr <dig> isoforms share obvious ribosome-related functions with their protein-coding host gene; they modify highly conserved residues on the 18s and 28s rrnas, respectively.
however, it is presently unclear whether the snm <dig> molecules represent functional entities, so their functional correlation with mfl, if any, remains to be proved.
nevertheless, the fine regulation of snm <dig> argues against the idea that they merely represent transcriptional noise.
even though these ncrnas may be involved in the same pathway as mfl, the possibility that they may have totally unrelated functions cannot presently be excluded.
in this case, it is conceivable that mfl may have been chosen as host merely to meet the need for a transcription rate high enough to produce a sufficient level of these molecules.
systematic mutagenic approaches are currently underway to address the biological role of these ncrnas, and to determine the degree, if any, to which they contribute to the mfl phenotype.
in any case, it is interesting to note that mammalian dkc <dig> genes have also been shown to host two intron-encoded h/aca snornas  <cit> , indicating that the coding/non-coding genetic architecture may represent an additional conserved feature shared by these highly related orthologues.
CONCLUSIONS
we report here that the drosophila gene encoding pseudouridine synthase has a complex coding/non-coding structure.
we have provided evidence that the use of different 3' end sites enables this gene not only to produce different mrnas but also to release distinct sets of small intron-encoded ncrnas, suggesting a potential novel role for overlapping 3'utrs.
our data also reveal that a minor variant mrna able to encode a distinct protein subform can be produced by this gene, suggesting that alternative splicing may have a role in regulating the expression of eukaryotic pseudouridine synthases.

 h/aca ribonucleoprotein particles in eukaryotes consist of four highly conserved core proteins and one molecule of h/aca small nucleolar rna, and most of them direct pseudouridylation of target rnas at specific sites. in this process, one of the core proteins acts as a pseudouridine synthase, while the h/aca snorna selects the residues to be isomerized via specific base-pairing. proteins catalyzing the conversion of uridines to pseudouridines belong to a highly conserved family, well-characterized examples of which include archaea, yeast and trypanosome cfb5p, drosophila mfl/nop60b, rat nap, and mouse and human dyskerin. in eukaryotes, these proteins accumulate in the nucleolus and participate in various cellular functions including processing and modification of ribosomal rna and maintenance of telomere integrity in mammals. genetic depletion experiments in different organisms have invariably shown that these proteins are essential for viability, indicating that they have important biological roles. the finding that human dyskerin is involved in two congenital diseases further supports this notion. mutations in these proteins are responsible for x-linked dyskeratosis congenita and for hoyeraal-hreidarsson syndrome, now recognized as a severe dc allelic variant. functional conservation of pseudouridine synthases is so remarkable that the archaeal acbf5p protein has recently been shown to assemble efficiently with a yeast h/aca snornp core component, nop and with human telomerase rna, which has a h/aca box motif. the biological role of rna pseudouridylation is still debated. it has been suggested that it contributes to rrna folding, rrnp assembly and ribosomal subunit assembly. subtle enhancing of ribosomal functions such as codon recognition has also been proposed. remarkably, recent data indicate that mutations in mammalian dyskerin impair translation from ires elements, thus specifically affecting cap-independent translation of a subset of mrnas. however, the role of h/aca snornps extends beyond ribosome biogenesis. in fact, although rrna is the most common modification target, spliceosomal snrnas or trnas can also be modified. furthermore, "orphan" snornas that lack complementarity with rrna or snrnas have also been described, and it is plausible that they target cellular rnas that remain unidentified. since proteins of the cbf5p family are essential for biogenesis and accumulation of h/aca snornas, mutations in them might trigger diverse effects. for example, different mutations of human dyskerin have been associated with reduced levels of distinct subsets of h/aca snornas, raising the possibility that some pathological aspects of dc may be related to the particular functions of the specifically-affected forms. indeed, the repertoire of functions attributed to members of the cbf family is wide and continually increasing; these genes may be involved in many biological processes. in yeast, cbf5p was first described as a low-affinity centromeric dna binding protein. subsequently, depletion of cbf5p was shown to cause nucleolar fragmentation and to disrupt the nucleolar localisation of trna. in mice, dkc alleles carrying hypomorphic mutations led with very high frequency to tumour development, indicating that the gene acts as a potent oncosuppressor. a novel role in snorna metabolism has recently been reported for the yeast and mammalian proteins: they are directly involved in the early steps of h/aca snornp assembly, since they are cotranscriptionally recruited at the 3' end of the nascent h/aca snornas. in trypanosomes, rnai-induced silencing of cbf has been shown not only to eliminate pseudouridylation on the spliced leader rna, but also to abolish its modification at the fourth cap- nucleotide. as a result of defects in the sl rna and decreased modification of the u small nuclear rnas, trans-splicing was inhibited at the first step of the reaction. considering the wide range of biological effects directed by members of the cbf gene family, it is plausible that this functional complexity might rely, at least in part, on the production of multiple transcripts with different properties. since a complex expression pattern is often observed for multifunctional genes in higher eukaryotes, we planned to analyse the molecular organization and transcriptional activity of the drosophila orthologue in greater detail, with the aim of better defining its coding properties and shedding new light on its complex functions. in previous studies, we isolated the gene encoding drosophila pseoudouridine synthase, called minifly, and showed that the use of alternative 3' utrs resulted in two main mrnas, and kb in length. these two species had different expression profiles, with the kb mrna constitutively expressed in both sexes throughout the life cycle, and the kb species mainly expressed in females and maternally transmitted to the developing embryos. however, the two transcripts had identical protein coding potentials and differed only in their 3' untranslated regions. as with genes encoding proteins involved in the synthesis, structure or function of the translational apparatus, mfl was shown to belong to the 5' top family. members of this family share a c residue at the + position followed by a 5– nt polypyrimidine tract in a short 5' non-translated region; their mrnas typically show growth-dependent translational regulation. we also showed that mfl hosts a snorna gene of the h/aca class within one of its introns. this gene, named snoh directs pseudouridylation of drosophila 18s rrna at position u, modification of which residue is conserved from yeast to man. in this paper we report that snoh is just one member of a variegated cluster of small ncrna genes hosted within mfl introns, revealing that this gene exhibits a much more complex overlapping coding/noncoding architecture than previously suspected. in addition, we show that mfl produces two novel coding transcripts. while one of these mrnas is characterized only by an extended 3'utr, the second is alternatively spliced, accumulates mainly in females, and encodes a rare variant protein that is predicted to have distinct functional properties. so far, mfl is the only eukaryotic pseudouridine synthase gene for which multiple mrnas have been reported, and the description of the variant protein and the various ncrnas it can encode may provide useful insights into the various functions of other members of this conserved family.  identification of two novel mfl coding transcripts in previous primer-extension experiments we detected and mapped a single mfl transcription start site. we also noticed that a very short upstream region of about bp separates mfl from the close, divergently-transcribed gene mrp on the basis of these observations, we considered that further transcript heterogeneity is more likely to derive from the alternative 3' ends. therefore, the transcriptional activity of the gene was first re-examined by 3'-race. based on the structures of the previously-described and kb mrnas, internal primers were derived from the sequence of exons and and used to search for additional poly sites, using poly+ rna extracted from male and female adult flies as template. in addition to the two expected bands, a longer product was obtained in each 3'-race reaction, revealing the presence of a poly site further downstream. these additional products were markedly more abundant in the female rna preparation, implying a sex-bias in the use of this novel 3' site. subsequent nucleotide analyses of these amplification products confirmed that mfl encodes a third mrna that differs from the and kb species only in the presence of three additional exons in a longer 3'utr. northern analysis of the poly+ rna preparations probed with the novel downstream exons revealed a transcript of about kb, expressed mainly in females. this is the expected length for an mrna species starting at the previously-mapped 5' site and terminating at the novel 3' end, further confirming that the, and kb mrnas are distinguished solely by 3'-end heterogeneity. we then searched for cis-acting control elements affecting mrna stability, location or translation within these overlapping 3'utr sequences. however, no functional motif currently annotated in the utresource database was identified in any of these regions. further experiments will therefore be required to establish whether these sequences include still-unidentified regulatory motifs. strikingly, northern analyses of poly+ rna revealed that a large polyadenylated transcript of about kb also derives from this downstream region. this species had already been detected in adult female or embryonic rna preparations by more upstream mfl genomic probes, but previous attempts to isolate cdna clones representing this form proved unsuccessful even after extensive screening of various cdna libraries. since in the present set of experiments we again failed to identify any product representing this rna, its structure remains elusive, so we cannot exclude the possibility that this species represents a variant transcript extending much further, or an unprocessed rna precursor. we next tried to connect the novel 3' exon cluster with more upstream exons by rt-pcr, with the aim of detecting alternative splicing events within the coding sequence of the gene. for this purpose, we used poly+ rna from male and female adult flies as template, and various combinations of forward primers positioned inside the coding region with reverse primers positioned along the kb-specific 3'utr exons. in each combination, primers derived from exons or always yielded a single amplification product, markedly more abundant in females; sequence analysis invariantly confirmed the structure of the kb mrna previously obtained by 3'-race. in contrast, two types of products were always obtained when a forward primer from exon in each combination of reverse primers, was used to reverse-amplify female rna. in addition to the expected band, a minor product of smaller size was noticed in each reaction. as an example, the result of an exon 2– rt-pcr amplification from female rna is reported in fig. 2a. in this case, in addition to the kb product expected from the kb mrna, an additional product of bp was observed. sequence analysis of the additional shorter products obtained in this set of experiments revealed an alternative splicing event that joins the 5' splice donor of exon to the 3' splice acceptor of exon to confirm the presence of this spliced mrna, the same 5' primer was used in combination with either a 3' primer spanning the 3– exon junction, able to detect only the alternatively spliced rna subform, or one spanning the 5– exon junction, able to detect the whole set of transcripts generated by the canonical splicing pattern comprising the kb constitutive mrna and the and kb maternal species. both primer pairs yielded positive amplification of a fragment with the expected size; moreover, both products were obtained at a significantly higher level in females, indicating that, as previously described for the kb and also for the kb species described above, the variant mrna exhibits a sex-preferential expression profile. a full-length cdna clone representing this novel mrna was obtained by combining 5' and 3' race experiments. gene-specific primers were designed to amplify the 5' end of the transcript and to give fragments partially overlapping with the 3'-race products. a short overlapping sequence in the 5' and 3' products allowed us to construct a virtually full length cdna by restriction digestion at the common bamh1site. the fragment obtained was about kb and its nucleotide sequence indicated that the novel mrna starts at the unique, previously-mapped transcription start site. intriguingly, this transcript is characterized by the skipping of five internal exons and the absence of any internal stop codon, indicating that it may encode a novel protein subform. given that the kb mrna species was barely detected in the northern analyses of poly+ rna preparations, we used quantitative real-time pcr to check its relative abundance in the poly+ rna preparations obtained from various sources: adult females, manually dissected adult ovaries and cultured schneider drosophila cells. the relative abundances of the variant and canonically-spliced mrnas were compared among the three samples after normalization against αtub84b expression. the kb mrna was significantly more abundant in ovaries than in whole adult females and its accumulation was at least nine-fold greater; the canonically-spliced mrna was also slightly enriched in this organ. in proliferating s cells, the alternatively-spliced species was markedly more abundant, whereas the level of the canonically-spliced mrnas was slightly reduced. structural properties and expression of the mflα novel protein subform the rare alternatively-spliced mfl mrna is predicted to encode a variant protein of amino acids, with a molecular mass of kda, which we named mflα. the amino acid sequence of this variant protein is shown in fig. 4a, aligned below that of mfl, identically encoded by the, and mrnas, and that of human dyskerin. mflα fully overlaps the mfl sequence at its amino-terminal region, where both proteins exhibit an identical tract of aa that includes the n-terminal nuclear location signal, and the two highly-conserved trubi and trubii motifs that share homology with bacterial and yeast trna pseudouridine synthases and are directly involved in the pseudouridylation process. however, mflα exhibits a unique c-terminal tract of residues that shows no significant homology with any known functional motif. instead, this region replaces a large carboxy-terminal portion of the mfl protein that includes at least three domains for which a functional role has been proposed. the first missing motif corresponds to the pua domain, an rna binding motif observed in several families of archaeal, bacterial and eukaryotic rna-modifying proteins including pseudouridine synthases and rrna methylases. this domain has also been found in bacterial and yeast glutamate kinases, as well as in families of eukaryotic proteins that are thought to act as translation factors. intriguingly, it has recently been shown to play a crucial role in archaeal snornp assembly, and to be necessary for acbf5p binding to guide rnas. the second missing domain is represented by a block of more than twenty residues with a central tyrosine that is identical in mfl and human dyskerin and is highly related to the uracil-binding pocket in uracil-dna glycosylases. finally, mflα also lacks a highly-charged lysine-rich carboxy-terminal region that contains an overlapping bipartite nls, raising the possibility that this variant protein may have a different subcellular distribution, or that it is less tightly retained in the nuclei. collectively, these distinctive features suggest that mflα may have a distinct role in at least some of the essential cellular activities of mfl. as a preliminary step to investigating this possibility, we attempted to assess the effective in vivo accumulation of this rare subform. on the basis of the observations indicating a higher abundance of the kb mrna in s cells, we selected this source for checking the presence of mflα protein by western blotting. rabbit polyclonal antibodies were raised against two peptides, both included in the common n-terminal region of mfl/mflα, and protein extracts from s cells were subjected to western analysis with these antibodies. in addition to the canonical mfl protein, a less abundant band of the molecular weight expected for mflα was detected, indicating that the alternatively-spliced mrna may encode a variant protein. a cluster of small ncrna genes is intron-encoded at the mfl variable 3' utrs to check mfl transcription further, we performed northern blotting of total rna preparations using genomic probes spanning the variable mfl 3' ends. the results showed that a set of small rna molecules, about nt long and highly abundant in females, also derive from this region. to map these molecules in greater detail, shorter probes having either exonic or intronic localisation were used to analyse rna preparations from adult flies of both sexes. probes derived from introns and all detected the kb species. they also detected a radioactive signal of about nt, as estimated by carefully assessing the rna length on 6% denaturing polyacrylamide gels and by mapping the 5' end by primer extension analysis. inspection of the mfl 3' genomic sequence indicated that introns and each host a copy of dmsnr, a snorna gene of the c/d family previously identified in the course of a genome-wide computational search. the strong cross-hybridization with rrna shown by these intronic probes is essentially explained by their perfect complementarity to the 28s rrna molecule, which is recognized by long d and d' antisense elements. as shown in the figure, probes spanning introns and detected rna molecules estimated at nt, again on the basis of 6% denaturing polyacrylamide gel electrophoresis and primer extension analysis of the 5' end, in addition to the and kb mrnas. nucleotide analysis of introns and revealed that each hosts a copy of a small ncrna gene, the sequence of which substantially overlaps that of the dmorc/d_ a-b molecules previously described by huang et al.. these molecules exhibit canonical d and d' boxes but have a degenerate c motif with a sequence varying between the two tandemly-repeated isoforms. the biological functions, if any, of these molecules remained to be firmly established, so they were classified as orphan c/d snornas. indeed, we noticed that they lack a nucleolar- or cajal body- specific location, so in accordance with widely-accepted nomenclature, we refer to them as small non-messenger rnas derived from polytene region. the two copies of snm share 85% overall sequence conservation and exhibit a bp internal segment of perfect identity. as judged by southern blotting and a computational search on the genome sequence, no other snm copy is present elsewhere in drosophila. organization of the ncrna gene cluster mapped at the mfl 3'-utrs conformed to the one-gene-per-intron rule widely observed in animal genomes. moreover, we noticed that the tandemly-clustered dmsnr genes are all located about nt upstream the 3' splice site of their host introns. in vertebrates, a position about nt upstream the 3' splice site is reportedly optimal for expression of intronic c/d box snornas, since this distance may furnish optimal synergy with splicing, favouring c/d box snornp assembly. the conserved position of dmsnr isoforms indicates that the rule observed by hirose and coworkers may also be operative in invertebrates. dmsnr genes, first described by our group, were reported in a subsequent genome-wide analysis to be generated, along with dmorc/d_ a-b molecules, by introns of a long, polyadenylated non-coding host transcript named duhg. however, identification of the additional mfl poly+ site in the present study provides clear evidence that dmsnr and dmorc/d_9/snm copies are all intron-encoded by this protein-coding gene. an intriguing functional consequence of the mfl coding/non-coding arrangement is that pre-mrnas ending at different alternative poly sites can release distinct sets of nested ncrnas. in fact, a pre-mrna molecule ending at the first poly site can exclusively produce the h/aca snorna h while one ending at the middle site may also release three dmsnr isoforms. a pre-mrna terminating at the most downstream site may instead produce three different types of small ncrnas, including snoh and all the dmsnr and snm isoforms. strikingly, the ability to release snm molecules is restricted to mfl transcripts ending at the last poly site, so that production of these ncrnas is predicted to be coupled with that of the and kb mrnas. in conclusion, alternative 3'-ends allow mfl to produce not only two distinct protein subforms, but also different ncrnas that may potentially contribute to its biological functions. expression and function of mfl intron-encoded ncrnas all the dmsnr isoforms possess canonical c, d and d' boxes, suggesting that they encode bifunctional snornas of the c/d family. long tracts of perfect complementarity to drosophila 28s rrna were found upstream of both the d and d' boxes. since c/d snornas invariably select the nucleotide positioned base pairs upstream of the d/d' box for methylation, these antisense elements were predicted to modify, respectively, the g and am residues, two methylation sites conserved between yeast and vertebrates. in fact, methylation at the g1083-equivalent residue is known to be guided by yeast snr and mammalian u snornas, while am modification is directed by yeast snr and by a still-unidentified mammalian snorna. methylated residues have not yet been experimentally mapped on drosophila rrna, so we attempted to detect effective modification at the predicted sites by reverse transcription of the specific rrna sequence at low dntp concentration, as described by maden. when this method is used, a specific reverse transcription stop occurs on a 2'-o-methylated nucleotide at low, but not at elevated, dntp concentrations. as shown in fig 6b, this experiment confirmed the presence of methylated nucleotides at both predicted positions. strikingly, one additional 2'-o-methylation was detected, at position gm of 28s rrna. modification at this site has not yet been described in other organisms and may be specific to drosophila rrna. since no other drosophila snorna that may be able to methylate the g and the am residues specifically has so far been described, modification of these sites strongly supports the functional role of the dmsnr molecules as rrna methylation guides. in contrast, the function of the snm molecules is hard to guess, and further experiments are required. to characterize further the small ncrnas originating from the mfl 3' region, a panel of total rna samples extracted from various stages of drosophila development or from the s cell line was subjected to northern blot analysis with probes specific for snoh dmsnr or snm these intron-encoded ncrna genes were all actively expressed in s cells, and throughout the drosophila life cycle they exhibited a very similar expression pattern. as shown in fig. the ncrnas accumulate constitutively throughout development, but their expression is quantitatively modulated, reaching the highest levels in young larvae and in adult females. it should be noted that the marked sex-bias in snoh and dmsnr expression is quite unusual for snornas. considering that these snornas are devoted to modifying, respectively, the 18s and 28s rrnas, it is plausible that the higher levels in females may essentially be due to the higher level of protein synthesis in the ovaries, which in adult females may constitute more than half the body weight. to address the function of dmsnr and snm further, we next investigated their intracellular distribution by in situ hybridization. given that these rnas accumulated at higher levels in larvae and in adult females, we selected the intestine as a larval organ and the ovary as an adult organ for these analyses. when specific digoxigenin-labelled antisense probes were used to analyse whole-mount intestine preparations, the dmsnr molecules were shown to be expressed in all cells. within ovarioles, these molecules were also expressed ubiquitously, being detected at all stages of oogenesis in both the nurse and the follicle cells. moreover, dmsnr molecules accumulate specifically in the nucleoli, as substantiated by observation of the large nuclei of the polyploid nurse cells. these cells develop unusual nucleoli comprising a shell of interconnected fibres around the nuclear periphery. as is evident from the figure, the dmsnr hybridization signal in the nurse cells is specifically concentrated in these nuclear peripheral structures, as expected for bona fide snornas. in contrast, snm expression in the ovarioles appeared to be restricted to late oogenesis, starting from stage; moreover, these rnas accumulated specifically in the nurse cells, not the follicle cells. within the nurse cells, the snm molecules appear to be concentrated in the nucleoplasm but are not located within the nucleoli. these ncrnas also show an intriguing expression pattern in the larval intestine, where in most microscopic fields they are detected only in subsets of cells that appear to be actively dividing. most cells expressing snm rnas are in fact yet-unseparated daughter cells just exiting from mitosis, or paired cells that plausibly derive from the same mitotic event, raising the possibility that expression of these molecules might be regulated during the cell cycle. an obvious conclusion from these in situ experiments is that spatial and temporal expression of dmsnr and smn genes are differentially regulated. it is possible that smn molecules may be independently transcribed from a specific alternative promoter, or that their biogenesis and/or stability may depend on the presence of specific factors. whatever the case, such fine regulation was unexpected and makes it unlikely that these small rnas merely represent transcriptional noise. to investigate this aspect further, we checked the phylogenetic conservation of smn genes by searching for their expression in drosophila-related species such as d. yakuba, d. virilis and d. ananassae. by northern blot experiments, we were able to detect these ncrnas only in the most closely-related species d. yakuba, which belongs to the melanogaster subgroup. in contrast, the dmsnr molecules were positively detected in all three species. a comprehensive picture of sequence conservation of the mfl 3' genomic region is presented in fig. which shows a diagram of multiple genome alignments obtained by the ucsc genome browser. peaks in the conservation plot can be observed for each of the 3'utr introns, but strong conservation among all the annotated genomes of the drosophila species occurs only for dmsnr isoforms. the weaker phylogenetic conservation displayed by snm suggests that these genes may have evolved more recently. however, evolutionary conservation may not be a reliable signature for functional ncrnas, since many regulatory ncrna evolve quickly and may co-evolve with their functional targets. discussion production of multiple transcripts with different coding/non-coding properties is known to contribute largely to the complexity of eukaryotic transcriptomes, and analysis of the full range of different transcripts that a gene can encode may provide important insight into its biological functions. although alternative mrna subforms have never been described for any member of the cbf5/mfl/dkc family, transcripts that have very low abundance, or are expressed in selected cell types or in response to specific stimuli, may have escaped analysis. indeed, rare products have important roles in many physiological processes and can often trigger crucial responses to developmental or growth stimuli. the detailed analyses of the mfl gene reported here show that its molecular organization is much more complex than previously suspected and its coding potential is correspondingly expanded. indeed, mfl is the only pseudouridine synthase gene for which extensive evidence of multiple transcripts has been reported so far. our results showed that the canonical mfl protein can be encoded by three mrnas distinguished solely by 3' end heterogeneity, two of them displaying a maternal pattern. these overlapping 3'-utrs may plausibly play a role in imposing different expression profiles on the mfl mrnas. in this regard, an intriguing recent suggestion is that translation of 5'-top mrnas may become less stringent with increasing 3'-utr length. consistent with this notion, it could be surmised that the diverse lengths of 3'-utrs in the, and kb mrnas may per se influence the efficiency of their translation under different growth conditions; moreover, the kb mrna is the species for which the most stringent translational regulation should be expected. the data reported in this paper also reveal that mfl encodes a novel alternatively-spliced protein subform, which represents the first example of a variant protein encoded by any member of the cbf gene family. the main distinctive trait of this protein, named mflα, is its unique c-terminus, where a short tract of amino acids replaces the large carboxy-terminal moiety of the major canonical protein, which includes the overlapping pua and tyr domains and the bipartite nls. given that the pua domain of archaeal acbf5p has recently been shown to be essential for binding the guide rnas, its lack is predicted to affect this functionally relevant feature, raising the possibility that mflα may participate in the formation of less efficient, or even inactive, h/aca snornps. if this were the case, expression of this subform might have an autoregulatory role in mfl expression. however, we cannot presently exclude the possibility that its specific c-terminus may allow mflα to interact with different efficiency or different specificity with distinct protein partners, eventually participating in the assembly of specific snornp subtypes. on the other hand, neither can we exclude the possibility that this subform has so far unknown functions, even unrelated to those of the major canonical protein. indeed, a further relevant distinctive trait of mflα is the absence of the c-terminal nls. this might plausibly allow a subtly modified modulation of the rate of nuclear transport, or of nuclear retention, or of the subnuclear distribution of the mflα subform in response to a various cellular stimuli. in this context, it is intriguing to note that deletion of the c-terminal lysine-rich cluster did not affect the nucleolar location of human dyskerin, but influenced the rate of transport into the nucleus, while truncation of the c-terminal basic domain of yeast cbf5p led to delay at the g2/m phase of the cell cycle. finally, we noticed that the mfl c-terminal moiety deleted in mflα is particularly rich in putative phosphorylation sites. since many cellular processes are controlled in a phosphorylation-and cell cycle-dependent manner, including protein synthesis and cell division, it is conceivable that mfl and mflα may respond in different ways to various growth conditions and cellular signals. an unexpected and distinctive peculiarity of mflα concerns its greater accumulation in adult females. although this sex-biased expression suggests that the subform is required during oogenesis and in the early stages of drosophila development, it would be premature to exclude the possibility that its expression may be identical in both sexes at different developmental stages, or that a sharply restricted or a transient expression profile may have hindered its detection in adult males. indeed, the data reported in this paper show that higher levels of expression in females are a general feature of mfl transcripts, either coding or non-coding, the only exception being the kb constitutive mrna, which has the same abundance in both sexes. this observation adds further strength to a recent report that included mfl/nop60b among the list of drosophila genes that are significantly more strongly expressed in purified female germline stem cells. it is also worth noting that the levels of all mfl transcripts peak at the major sites of cell growth and division during the drosophila life cycle – late embryos, larvae and adult ovaries – making it plausible that gene expression might be linked to the rate of cell growth and proliferation. this hypothesis is compatible with the essential role in ribosome biogenesis and with recent microarray analyses that indicated mfl/nop60b to be a major target of d-myc induction. in this light, the different levels of mflα expression between male and female adult flies may only be coincidental, simply reflecting the different rates of protein synthesis in adult flies of different sexes. although further experiments are required to elucidate the molecular mechanisms that regulate mfl expression, the intriguing pattern displayed by snm molecules in proliferating intestine cells is also in good agreement with this view. the complex molecular organization described here for mfl may be of general relevance, possibly unravelling aspects common to other orthologues. although no variant dkc transcript has been characterized thus far in mammals, it is intriguing that deletion and splice site mutations in the last exon of the gene have been found in dc patients, and that a small deletion covering only the last dkc exon proved to be lethal in mouse knock-out experiments. together, these observations indicate that the 3' portion of the gene is also highly biologically relevant in mammals. moreover, dkc -deleted alleles triggered early lethality only when they occurred on the maternal allele, revealing the existence of a maternal effect in the transmission of dc disease. it is tempting to speculate that, similarly to mfl, mammalian dkc genes may encode still-undetected transcripts, the expression of which may account for this sex-specific effect. our data also show that mfl introns harbour a variegated cluster of small ncrna genes comprising an h/aca snorna gene, four copies of the c/d snorna dmsnr and two copies of the small ncrna of unknown function. the structure of this dense cluster conforms to the one ncrna gene per intron rule widely observed in animal genomes, and strongly implies that several duplication events have occurred during its evolution. this coding/non-coding genetic arrangement has obvious regulatory potential: to coordinate the expression of nested ncrnas with the protein products of the gene. common functional roles are often shared by host genes and intron-encoded ncrnas. in the case of mfl, the snorna h and dmsnr isoforms share obvious ribosome-related functions with their protein-coding host gene; they modify highly conserved residues on the 18s and 28s rrnas, respectively. however, it is presently unclear whether the snm molecules represent functional entities, so their functional correlation with mfl, if any, remains to be proved. nevertheless, the fine regulation of snm argues against the idea that they merely represent transcriptional noise. even though these ncrnas may be involved in the same pathway as mfl, the possibility that they may have totally unrelated functions cannot presently be excluded. in this case, it is conceivable that mfl may have been chosen as host merely to meet the need for a transcription rate high enough to produce a sufficient level of these molecules. systematic mutagenic approaches are currently underway to address the biological role of these ncrnas, and to determine the degree, if any, to which they contribute to the mfl phenotype. in any case, it is interesting to note that mammalian dkc genes have also been shown to host two intron-encoded h/aca snornas, indicating that the coding/non-coding genetic architecture may represent an additional conserved feature shared by these highly related orthologues.  we report here that the drosophila gene encoding pseudouridine synthase has a complex coding/non-coding structure. we have provided evidence that the use of different 3' end sites enables this gene not only to produce different mrnas but also to release distinct sets of small intron-encoded ncrnas, suggesting a potential novel role for overlapping 3'utrs. our data also reveal that a minor variant mrna able to encode a distinct protein subform can be produced by this gene, suggesting that alternative splicing may have a role in regulating the expression of eukaryotic pseudouridine synthases. 
in addition to catalyzing pseudouridylation of target rnas, they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to telomere maintenance. this gene exhibits a complex and interlaced organization, and its genetic information may be expressed as different protein subforms and/or ncrnas that may potentially contribute to its biological functions. to investigate further the molecular mechanisms underlying the expression of pseudouridine synthase genes, we analyzed the transcriptional activity of the drosophila member of this family in great detail. we also report that a cluster comprising four isoforms of a c/d box snorna and two highly related copies of a small ncrna gene of unknown function is intron-encoded at the gene-variable 3'utrs. intriguingly, accumulation of all these intron-encoded rnas was found to be sex-biased and quantitatively modulated throughout development and, within the ovaries, the ncrnas of unknown function were found not ubiquitously expressed. the rare spliced variant is detected mainly in females and is predicted to have distinct functional properties. our results expand the repertoire of coding/non-coding transcripts derived from the gene encoding drosophila pseudouridine synthase. the drosophila gene for pseudouridine synthase, minifly/nop60b, encodes two novel mrnas ending at a downstream poly site. because this arrangement, the alternative 3' ends allow mfl not only to produce two distinct protein subforms, but also to release different ncrnas. in eukaryotic cells, each molecule of h/aca small nucleolar rna assembles with four evolutionarily conserved core proteins to compose a specific ribonucleoprotein particle. one of the four core components has pseudouridine synthase activity and catalyzes the conversion of a selected uridine to pseudouridine. members of the pseudouridine synthase family are highly conserved. one species is characterized only by an extended 3'-untranslated region, while a minor mrna encodes a variant protein that represents the first example of an alternative subform described for any member of the family to date.
 in eukaryotic cells, each molecule of h/aca small nucleolar rna assembles with four evolutionarily conserved core proteins to compose a specific ribonucleoprotein particle. one of the four core components has pseudouridine synthase activity and catalyzes the conversion of a selected uridine to pseudouridine. members of the pseudouridine synthase family are highly conserved. in addition to catalyzing pseudouridylation of target rnas, they carry out a variety of essential functions related to ribosome biogenesis and, in mammals, to telomere maintenance. to investigate further the molecular mechanisms underlying the expression of pseudouridine synthase genes, we analyzed the transcriptional activity of the drosophila member of this family in great detail.  the drosophila gene for pseudouridine synthase, minifly/nop60b, encodes two novel mrnas ending at a downstream poly site. one species is characterized only by an extended 3'-untranslated region, while a minor mrna encodes a variant protein that represents the first example of an alternative subform described for any member of the family to date. the rare spliced variant is detected mainly in females and is predicted to have distinct functional properties. we also report that a cluster comprising four isoforms of a c/d box snorna and two highly related copies of a small ncrna gene of unknown function is intron-encoded at the gene-variable 3'utrs. because this arrangement, the alternative 3' ends allow mfl not only to produce two distinct protein subforms, but also to release different ncrnas. intriguingly, accumulation of all these intron-encoded rnas was found to be sex-biased and quantitatively modulated throughout development and, within the ovaries, the ncrnas of unknown function were found not ubiquitously expressed.  our results expand the repertoire of coding/non-coding transcripts derived from the gene encoding drosophila pseudouridine synthase. this gene exhibits a complex and interlaced organization, and its genetic information may be expressed as different protein subforms and/or ncrnas that may potentially contribute to its biological functions. 