both RpL22 and RpL22-like are present within the same ribosome), it is reasonable to position RpL22-like similar- ly, yet propose that its interactions may differ. Alternative splicing of rpL22-like through intron retention This study demonstrated that alternative splicing of rpL22-like generates two structurally distinct mRNA isoforms that are enriched within testes. A rare novel mRNA transcript called rpL22-like short results from splicing of an intron that is retained within the more abundant, full-length rpL22-like. Basal levels of rpL22-like short mRNA are detectable by RT-PCR within heads and ovaries suggesting that the alternative splicing machinery is not limited to testis; however, the process may be subject to speciﬁc regulation within the testis where rpL22-like short mRNA levels are compara- tively more abundant. It is unknown if alternative splicing of rpL22-like occurs in all stages of spermatogenesis or if splicing is cell-type speciﬁc. The low abundance of rpL22-like short mRNA relative to the full-length variant may be consistent with a specialized role in a subset of spermatocytes. Stage-speciﬁc gene expression during ﬂy spermatogenesis has been the subject of intense study, showing that some testis-speciﬁc genes are activated in primary spermatocytes while others (comet and cup genes) are transcribed post-meiotically, even within mid-to-late stage elongating spermatids in Drosophila [reviewed by ref. (28)]. Although unique splicing phenomena of this type have not been described in this system, stage-speciﬁc splicing in sperm- atogenesis remains an intriguing possibility, Intron retention is among the rarest forms of splicing in vertebrates and invertebrates; yet, it is the most prevalent type of alternative splicing found in protozoa, fungi and plants [reviewed by ref. (29)]. This phenomenon has been described in several other Drosophila genes [e.g. Suppressor-of-white-apricot (30), c-cam3 (31), Sxl (32), erect wing (33), transformer-2 (34), nuclear export factor 1 (35)] and the consequences of retaining an intron can have profound effects on gene expression leading to an unequal accumulation of mRNA variants. For rpL22- like, the major form retains the intron. Sakabe and de Souza (36) identiﬁed several features that support a higher incidence of intron retention: weak splice sites, genes with overall short intron lengths and higher expression levels, and speciﬁc densities of splicing regula- tory elements. Non-canonical splice sites were eliminated from the analysis, although it was apparent that bona ﬁde examples of splicing using non-canonical splice site recog- nition were present in their human gene data sets. Other studies have further evaluated non-canonical splice site usage in a number of organisms (e.g. 25,37,38). Sheth et al. (38) categorized sub-types of splice sites based on U2- and U12-dependent spliceosomes and described additional rare splice site types. In at least one case in Drosophila, an intron within the rudimentary gene is deﬁned by a CT donor SS (Flybase.org). Although originally noted in Mount et al. (37) that the ﬂy perB intron E has a 30SS of CG, the most recent version of Nucleic Acids Research, 2011, Vol. 39, No. 7 2713 Flybase (2010_08) has revised this proposal to reﬂect a canonical splice site. The proposed retained intron is deﬁned by a set of non-canonical splice site signals (50SS: CT; 30SS: CG) that may impact not only the mechanism of splicing, but also the kinetics of splicing regulation. It is noted that both 50 and 30 splice site sequence motifs are weak (MAXENT scores: 15.93 and 15.67, respectively) compared to the 50 and 30 scores for the upstream intron in rpL22-like (MAXENT scores: 8.57 and 7.23, respect- ively) when analysed using a human splice site model to predict splice site strength [MaxEntScan: (39)]. These data are consistent with an intron retention model (36) and support the conclusion that the splicing rates for the upstream intron and the retained intron are different. Intron retention often results in premature stop codon insertion, thereby directing the alternative transcript to the nonsense mediated decay (NMD) pathway (40). The absence of premature termination codons in the short variant sequence likely eliminates this variant from NMD, and favours the interpretation that the short variant has functional signiﬁcance by generating a novel protein, as proposed in this study. Using D. melanogaster nucleotide and amino acid sequences for rpL22- like in a BLAST search for homolo- gous sequences in other sequenced Drosophila species, we show that ﬁve other species (D. sechellia, D. simulans, D. erecta, D. ananassae and D. yakuba) contain an orthologous gene (Supplementary Figures S5 and S6). The other six species (D. pseudoobscura, D. persimilis, D. wilistoni, D. mojavensis, D. virilis and D. grimshawi) lack an rpL22-like orthologue. Within species that contain an rpL22-like orthologue, production of an rpL22-like short orthologue is also theoretically possible, generated by alternative splicing using conserved non-canonical sequences (50SS: CT; 30SS: CG) at the retained intron/exon boundaries (Supplementary Figures S5 and S6). Evolutionary conservation of the al- ternative splicing pattern in the melanogaster lineage would lend further support that the alternative tran- script encodes a functional product with a structure that is generally conserved in all members of the melanogaster group except D. ananassae (see sequence alignment Supplementray Figure S6). Protein structural diversity generated through alternative splicing of rpL22-like Alternative splicing contributes to the enormous amount of protein diversity observed within eukaryotic cells. The rarer mRNA isoform (rpL22-like short) encodes a protein in which the majority of the C-terminal RP signature has been eliminated. Most of the structure of the short protein variant is comprised of the majority of the divergent N-terminal domain. Functional residues previously identiﬁed in RpL22 that are required for nuclear and nu- cleolar localization as well as RNA binding (20) are absent from this short protein variant, possibly restricting its subcellular compartmentalization and RNA binding ability. Alternatively, other regions of the protein may have redundant functions that would replace missing