Published online 7 December 2010 Nucleic Acids Research, 2011, Vol. 39, No. 7 2701–2716 doi:10.1093/nar/gkq1218 Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced Michael G. Kearse, Alex S. Chen and Vassie C. Ware* Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA Received July 22, 2010; Revised November 9, 2010; Accepted November 10, 2010 ABSTRACT Several ribosomal protein families contain paralogues whose roles may be equivalent or specialized to include extra-ribosomal functions. RpL22e family members rpL22 and rpL22-like are differentially expressed in Drosophila melanogaster: rpL22-like mRNA is gonad specific whereas rpL22 is expressed ubiquitously, suggesting distinctive paralogue functions. To determine if RpL22-like has a divergent role in gonads, rpL22-like expres- sion was analysed by qRT-PCR and western blots, respectively, showing enrichment of rpL22-like mRNA and a 34 kDa (predicted) protein in testis, but not in ovary. Immunohistochemistry of the reproductive tract corroborated testis-specific expression. RpL22-like detection in 80S/polysome fractions from males establishes a role for this tissue-specific paralogue as a ribosomal compo- nent. Unpredictably, expression profiles revealed a low abundant, alternative mRNA variant (designated ‘rpL22-like short’) that would encode a novel protein lacking the C-terminal ribosomal protein signature but retaining part of the N-terminal domain. This variant results from splicing of a retained intron (defined by non-canonical splice sites) within rpL22-like mRNA. Polysome association and detec- tion of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing of rpL22-like generates structurally distinct protein products: ribosomal component RpL22-like and a novel protein with a role distinct from RpL22-like. INTRODUCTION In several ribosomal protein (RP) gene families [most not- able in certain yeast species and plant systems—reviewed by ref. (1)], paralogous proteins exist, presumably derived from duplication events in the evolutionary history of the gene. Paralogous RPs may have functionally redundant roles within the ribosome, or in some instances, their roles may be specialized in ribosome biogenesis or translation, contributing to heterogeneity within the ribosome cycle [e.g. (2)]. Alternatively, specialized roles for paralogous RPs may include extra-ribosomal or extra-translational functions [see review by Warner and McIntosh (3) for some discussion on this issue]. Specialized roles may be indicated particularly if a paralogue is expressed in a cell-, tissue- or developmental stage-speciﬁc manner. Recent studies in Saccharomyces cerevisiae have revised the previously held view that many RP paralogues dually expressed in that species, are functionally equivalent (4). Instead, some paralogues are specialized for differential functions or cellular locations (4,5), leading Komili et al. (4) to propose a ‘ribosome code’ that regulates translation of speciﬁc mRNAs in different physiological states. Although less than that reported in yeast and plant systems, tissue-speciﬁc ribosome heterogeneity due to assembly of RP structural variants into ribosomes has also recently been reported in rodent mammary gland and liver for RpL22-like1 and in testis for RpL10- and RpL39-like (2). In Drosophila melanogaster, RpL22 and RpL22-like are members of the conserved RpL22e family speciﬁc to eu- karyotes. Unlike most ﬂy RP paralogues that display between 65% and 100% amino acid identity (6), RpL22 and RpL22-like are instead only 37% identical (6), sug- gesting considerable ‘opportunity’ for disparate functions between family members. RpL22 family members in Drosophila also exhibit unique structural features at the N-terminus compared to orthologues in other species. Fly RpL22e family members contain an N-terminal extension of unknown function that is homologous to the C-terminal end of histone H1 [previously described only for RpL23a and RpL22 by ref. (7)]. Structural divergence between RpL22 and RpL22-like is most prominent within the N-terminal extension. Over time the novel domain *To whom correspondence should be addressed. Tel: +610 758 3690; Fax: +610 758 4004; Email: vcw0@lehigh.edu The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.