2714 Nucleic Acids Research, 2011, Vol. 39, No. 7 functional segments. In fact, a computational prediction using ELM (http://elm.eu.org/) highlights a bipartite variant of the classical NLS containing basic residues at RpL22-like short amino acid positions 90–111 (and also predicted for RpL22-like) that may prove functional for nuclear import and subnuclear compartmentalization. Collectively, our data support the conclusion that rpL22-like encodes not only a RP, but an extra-RP as well. In silico analyses may provide clues about a putative extra-ribosomal function for the short protein. Although no speciﬁc DNA-binding motifs are apparent (41), 51 of 123 amino acid residues have DNA binding capacity, with 18 of those residues clustered within the ﬁrst 24 amino acid at the very N-terminal end (42,43). Given this prediction (and its limited structural similarity to histone H1), we speculate that the short protein variant may interact with testis chromatin and have a role in chro- matin repackaging and condensation during the transition from a nucleosome-based to a protamine-based conﬁgur- ation, as occurs in maturing sperm cells during spermiogenesis [e.g. reviewed by ref. (44)]. Perspectives on RpL22e paralogue function and evolution Paralogous members of the RpL22e family have been described in other animal genomes [e.g. rpL22-like1 in Mus musculus (NP_080793.1), Danio rerio (NP_001038800), Xenopus tropicalis (Q5I0R6), Homo sapiens (AAH62731), Rattus norvegicus (NP_001102018.1)] and their tissue-speciﬁc expression often varies considerably compared with the ﬂy pattern described here (45). Relatively little is known about func- tional redundancy or speciﬁcity of other rpL22e paralogues in other species; however, in at least one case, an rpL22 knockout mouse only exhibited a mild phenotype in T cell development, but was otherwise viable and fertile (46), suggesting that mouse paralogue rpL22-like1 could rescue critical functions lost by rpL22 disruption. Proteins of the RpL22e family in Caenorhabditis elegans [RpL22; (47)] and Drosophila are essential [RpL22: (48,49)]. However, this is not the case for RpL22 in yeast (50) or in mice as discussed above (46). Flybase reports that a P-element chromosomal insertion located 150 nts upstream of rpL22-like is lethal in ﬂy development, suggesting that the rpL22-like gene is also essential. The nature of the essential RpL22-like function within the ﬂy is intriguing, since one would predict that disruption of RP function in the testis would affect male fertility, and not viability. This suggests that rpL22-like has an essential role (ribosomal or non-ribosomal) in another tissue(s) (head or eye) at some stage in ﬂy development. In this case, it appears that RpL22 cannot replace the function lost by RpL22-like disruption, either because of a lack of co-expression in the appropriate tissue in time and space or because the role of RpL22-like is unique. Similarly, RpL22-like is unable to replace functions lost by RpL22 disruption, correspondingly suggesting that essential func- tions prescribed by each protein are not redundant. Do the essential roles of these paralogues include non-ribosomal roles? The presence of anti-L22-like and anti-L22 immunoreactive products in high-MW complexes within eye tissue and the testis, respectively, raises the possibility that each paralogue may be post-translationally modiﬁed or bound within detergent-resistant complexes and function in a role(s) that is distinct from its ribosomal role, since neither high-MW species was found in association with ribosomes. From an evolutionary perspective, gene duplication is the likely mechanism by which paralogous genes arise and generate tissue- or lineage-speciﬁc genes [e.g. (51)]. Several inferences about RpL22e family evolutionary history become apparent from genomic analyses of the Drosophilidae. We and others (8) propose that rpL22 is the ancestral gene that was duplicated in a series of complex events whose extant outcome is the ubiquitous expression of rpL22 and cell lineage-speciﬁc expression of rpL22-like. The rpL22-like gene is only found in the melanogaster group. One hypothesis addressing why obscura lineage genomes lack the rpL22-like gene requires that gene duplication occurred in the melanogaster group, allowing for essential functions to be shared between paralogues. Over time gene divergence may have directed changes in paralogue function. The absence of the rpL22-like gene from the obscura lineage may indicate that ‘essential’ functions were retained in the an- cestral rpL22 gene or are provided by another gene(s). No doubt comparative analyses of paralogue expression and function in different Drosophila species will be instrumen- tal in providing further insights into the evolutionary history and functional diversity displayed within the RpL22e family. ACCESSION NUMBERS GenBank accession HM756190 and HQ190956. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Dr Anna Gumpert for microscopy assistance, Dr Lynne Cassimeris for Alexa 2 Ab, Kandiss Schrader for S2 cells, Lee Graham (Lehigh) and Todd Laverty (HHMI-JFRC) for ﬂies, Dr Brian Chen for advice about proteomics tools, and Maria Brace for ﬁgure preparation assistance. Dr Michael Kuchka and Dr Barry Bean are acknowledged for stimulating discussions about the work reported here. Dr Michael Kuchka and Dr Lee Hughes are acknowledged for comments about the manuscript. FUNDING Lehigh University Faculty Research Grants (607233, 607277 to V.W., partial); Pennsylvania Department of Health (Block Grant). The work described here is in partial fulﬁlment of the requirements for the PhD degree