Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Nucleic Acids Research, Apr 2011

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 expression 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 component. 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 detection 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.

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Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced

Michael G. Kearse 0 Alex S. Chen 0 Vassie C. Ware 0 0 Department of Biological Sciences, Lehigh University , Bethlehem, PA 18015, USA 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 expression 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 component. 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 detection 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. - 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 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 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 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 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 in D. melanogaster, their expression patterns are also dissimilar. Transcripts for rpL22 are ubiquitously expressed. Previous studies have revealed rpL22-like mRNA expression in embryonic gonads, adult ovary and germline stem cells by in situ hybridization or RT-PCR (810). Recent microarray analyses showed enrichment of rpL22-like in 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 of rpL22-like expression suggests that 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 (...truncated)


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Michael G. Kearse, Alex S. Chen, Vassie C. Ware. Expression of ribosomal protein L22e family members in Drosophila melanogaster: rpL22-like is differentially expressed and alternatively spliced, Nucleic Acids Research, 2011, pp. 2701-2716, 39/7, DOI: 10.1093/nar/gkq1218