Both RpL22 and RpL22-like are detected in 80S ribosome and polysome fractions, indicating that both proteins are stably associated with ribosomes

Both RpL22 and RpL22-like are detected in 80S ribosome and polysome fractions, indicating that both proteins are stably associated with ribosomes. 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) withinrpL22-likemRNA. Polysome association and detection of a low abundant 13.5 kDa (predicted) protein in testis extracts suggests variant mRNA translation. Collectively, our data show that alternative splicing ofrpL22-likegenerates 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 notable in certain yeast species and plant systemsreviewed 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 D-Ribose 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 Rabbit Polyclonal to UBE1L particularly if a paralogue is expressed in a cell-, tissue- or developmental stage-specific manner. Recent studies inSaccharomyces cerevisiaehave 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 Komiliet 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). InDrosophila melanogaster, RpL22 and RpL22-like D-Ribose are members of the conserved RpL22e family specific to eukaryotes. 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), suggesting considerable opportunity for disparate functions between family members. RpL22 family members inDrosophilaalso 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 D-Ribose N-terminal extension. Over time the novel domain may have specified new functions for these proteins in addition to their functions in the ribosome cycle. In addition to considerable amino acids divergence between these paralogues inD. melanogaster, their expression patterns are also dissimilar. Transcripts forrpL22are ubiquitously expressed. Previous studies have revealedrpL22-likemRNA expression in embryonic gonads, adult ovary and germline stem cells byin situhybridization or RT-PCR (810). Recent microarray analyses showed enrichment ofrpL22-likein adult testis, but not in adult ovary [FlyAtlas; (11)]. Shotgun mass spectrometric data support the existence of RpL22-like protein in fly embryos (www.ebi.ac.uk/pride/Q8T3X3), but no protein expression data for other developmental stages and/or specific tissues have been established. Tissue-specificity ofrpL22-likeexpression suggests that D-Ribose RpL22-like may have a distinct role compared to its paralogue RpL22, at least in the embryonic gonad. Although its position on the 60S subunit has recently been mapped by cryoEM to the base of the subunit on the most recently published 80S ribosome model (12), the cellular role for RpL22 has not been completely characterized (13). Interestingly, partially reconstituted ribosomes that lack RpL22 are still translation competent, suggesting that the protein may have a regulatory or non-ribosomal role (13), or alternatively, function under different physiological conditions. InDrosophila, additional roles and interactions for RpL22 have been proposed [based on high-throughput yeast two hybrid screens assembled in the Drosophila Interactions Database version 2010_10 (DroID:http://www.droidb.org)], awaiting further characterization. Among these interactions are several putative extra-ribosomal roles for RpL22, including interactions with a transcriptional repressor complex in Kc cells (14) and with nuclear enzyme poly-ADP ribose polymerase [mediated through the N-terminal histone H1-like domain (7)], for example. Based on C-terminal homology to RpL22 and its tissue-specific expression pattern, it is reasonable.