2708 Nucleic Acids Research, 2011, Vol. 39, No. 7 Figure 5. Immunoﬂuorescent staining of RpL22 and RpL22-like in adult male reproductive tract using paralogue-speciﬁc Abs. Extruded sperm (ES) were released from the testes during dissection. Spermatogenesis is initiated at the apical tip of the testis (T) and progresses through the testis coils. The most mature sperm are, therefore, located distal to the apical end of the testis. Mature sperm pass from the testis into the seminal vesicle (SV). Seminal ﬂuid is added to sperm from the accessory gland (AG), and sperm are released through the ejaculatory duct (ED). RpL22 (red) is ubiquitously expressed in the reproductive system. RpL22-like (green) is expressed exclusively within the testis and within sperm cells. DNA is visualized by DAPI staining (blue). To rule out non-speciﬁc or off-target ampliﬁcation, the lower MW amplicon was cloned and sequenced. Sequence analysis of 29 different clones (2: embryo; 2: larvae; 6: testis; 19: ovary) derived from PCR products using different RNA samples and primer sets (Table 1) conﬁrmed that the lower MW amplicon was derived from the rpL22-like gene and therefore presumed to be a previously unidentiﬁed spliced variant of rpL22-like mRNA (Figure 2). We have named this novel mRNA product ‘rpL22-like short’ to reﬂect its truncated structure. Its deduced sequence of 123 amino acid would consist of the ﬂy-speciﬁc histone H1-like N-terminal ex- tension fused in frame to the last 10 amino acid in the C-terminus (Figure 1B). The proposed protein sequence lacks 189 amino acid that comprises the majority of the conserved RpL22e family signature at the C-terminal end. Alignment of the coding sequences for rpL22-like short (GenBank accession no. HM756190) and full-length rpL22-like (GenBank accession no. HQ190956) revealed a surprising ﬁnding that the proposed splice sites sur- rounding the uncharacterized intron (0.567 kb) were non-canonical in sequence [50 splice site (SS): CT; 30 SS: CG] compared to typical sequences found in most introns (50 SS: GT; 30 SS: AG), including the annotated intron within the rpL22-like gene (Figure 7). Non-canonical splice sites are indeed rare, but have been described in other eukaryotic genes as well [e.g. (25)]. In all 29 rpL22-like short sequenced clones, proposed 50 splice donor and 30 splice acceptor sites were identical to those shown in Figure 7, strongly supporting new intron–exon deﬁnitions within rpL22-like. Template switching artifacts can occur in some cases where RT-PCR is performed on templates that have direct repeats ﬂanking a proposed intron, thereby giving false signals of truncated, alternatively spliced products (26). Although direct repeats do not ﬂank the proposed retained intron in this case, we used additional RT-PCR analyses with primers that speciﬁcally hybridize to proposed exon–exon junctions and overlap the junction by 4 nt, expecting that an rpL22-like short amplicon would be produced (using stringent conditions) only if the proposed exon–exon junction was present. In all cases, RT-PCR amplicons were consistent with expected