2702 Nucleic Acids Research, 2011, Vol. 39, No. 7 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 expres- sion 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 hybridiza- tion or RT-PCR (8–10). Recent microarray analyses showed enrichment of rpL22-like in adult testis, but not in adult ovary [FlyAtlas; (11)]. Shotgun mass spectromet- ric 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 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. In Drosophila, 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 to hypothesize that RpL22-like has a gonad-speciﬁc ribosomal function, although other functions cannot be excluded. In fact, several protein–protein interactions are also catalogued for RpL22-like in the Drosophila Interactions Database and none overlap with those proposed for RpL22, including those that are likely to be non-ribosomal in nature. Together, this information suggests that RpL22 and RpL22-like have distinct functions, either within the ribosomal cycle and/or in non-ribosomal pathways. That one of the functions of RpL22-like is as a ribosomal com- ponent had not been previously investigated prior to this study. Such developmental or tissue-speciﬁc regulation of a putative RP is not widely known in animal systems and is more commonplace in plants (1). To explore the possibility that RpL22-like functions as a tissue-speciﬁc ribosomal component, we ﬁrst reﬁned its developmental and tissue-speciﬁc expression pattern to facilitate its biochemical characterization. By quantitative (q) RT-PCR, we determined that rpL22-like mRNA is highly enriched in adult testes compared to ovaries. Using paralogue-speciﬁc antibodies (Abs) in western blots, we detected a highly abundant protein of the predicted molecular weight (MW) for RpL22-like in testes. A higher MW immunoreactive species was also detected in ﬂy heads. Immunohistochemical (IHC) analysis of the male reproductive tract shows that RpL22-like is exclusively found within testes and not within seminal vesicles or accessory glands. We further demonstrate that RpL22-like is a ribosomal component (80S and polysomes), suggesting its incorporation into actively translating ribosomes. These studies also led to a novel ﬁnding that rpL22-like is alternatively spliced using non-canonical splice sites to remove an intron that generates a short form designated rpL22-like short, found in lower abundance than the full- length mRNA isoform. Surprisingly, the most abundant rpL22-like mRNA isoform retains the previously uncharacterized intron. rpL22-like short mRNA would encode a protein consisting nearly exclusively of amino acid residues in the N-terminal domain of RpL22-like fused in frame to residues at the very end of the C-terminus, thereby eliminating the majority of the conserved L22e RP signature. Detection of rpL22-like short mRNA on polysomes and the presence of a low abundant protein of the predicted MW in testis extracts suggest that the spliced variant may be translated. This study provides the ﬁrst experimental conﬁrmation that RpL22-like is a ribosomal component that is enriched in testis, and that its gene through alternative splicing may also encode a novel protein (RpL22-like short) with a novel non-ribosomal function based on its predicted amino acid structure. METHODS Fly stocks Wild-type Canton S D. melanogaster were used for polysome proﬁling (a kind gift from Todd Laverty, HHMI-JFRC). All other experiments used wild-type Oregon R D. melanogaster obtained from Carolina Biological. Primers For a list of primers and oligonucleotides (Integrated DNA Technologies) used in this study, see Table 1. Antibodies The following peptide sequences were obtained from FlyBase (15) and used for polyclonal Ab generation from GenScript: RpL22: FRISSNDDEDDDAE; RpL22-like: ADDNGGKTFA. Polyclonal Abs were protein A puriﬁed from rabbit and mouse, respectively. HRP- conjugated secondary Abs (Promega) were used in western analysis. Antibody speciﬁcity was tested using bac- terially expressed recombinant RpL22 and RpL22-like (Supplementary Figure S2). Preimmune sera from rabbit and mouse were used to conﬁrm the absence of immunoreactive proteins in ﬂy extracts (Supplementary Figure S2). Anti-mouse Alexa 488 Fluor and anti-rabbit Alexa 568 Fluor secondary Abs (Invitrogen) were used for IHC.