2710 Nucleic Acids Research, 2011, Vol. 39, No. 7 Figure 7. RpL22-like coding region showing novel splice site junctions for rpL22 like-short. Exon sequences (capitalized) and intronic sequences (lowercase) were derived from Flybase (FBgn0034837). The coding region of rpL22-like is shown in blue caps. The previously annotated intron (FlyBase FB2010_06) is shown in green lowercase. The novel intron is represented in bold with non-canonical splice sites underlined (50SS: CT, 30SS: CG). The rpL22-like short sequence (GenBank accession no. HM756190) was derived from sequencing multiple (29 total) cloned cDNAs from RT-PCR analyses (Figure 2). Red and purple arrows in the splicing diagram represent primer pairs used in RT-PCR analyses in Figure 8A and Supplementary Figure S1. such if rpL22-like mRNA is alternatively spliced and the intron is stable. Alternatively, this species may represent a speciﬁc degradation product derived from rpL22-like mRNA. In general, detection of introns would be rare, unless the proposed intron encodes a stable, functional small RNA, as has been shown for small nucleolar RNAs (Psi18S-531; Psi28S-2179; Flybase) encoded by rpL22 introns. When the same embryo RNA blot was stripped and re-probed with ﬂanking probes, the pattern of hybridization differed from the intron-speciﬁc hybridiza- tion pattern in that the 0.7 and 0.5 kb species were clearly present as previously noted in the developmental RNA blot. The presumptive intron species was not apparent in this blot using ﬂanking probes. An unidenti- ﬁed RNA species of 0.9 kb is also prominent, and may be faintly represented in all stages in the develop- mental blot (Figure 8B). Importantly, the northern blot data demonstrate the presence of smaller RNA species not previously predicted for rpL22-like based on genome annotation in Flybase and may represent al- ternatively spliced mRNA variants although we cannot conclusively discount the possibility that smaller RNA species might represent speciﬁc rpL22-like mRNA degrad- ation products detected with our probes. When northern blot data, together with RT-PCR ampliﬁcation data using in vivo and in vitro RNA templates are considered, we favour the conclusion that rpL22-like short mRNA is a bona ﬁde transcript produced by alternative splicing of the retained intron found within rpL22-like mRNA. rpL22-like short mRNA is associated with polysomes and may be translated Initial western analysis of testis extracts (at protein concentrations sufﬁcient to detect RpL22-like) did not detect a protein product of the expected MW for RpL22-like short (Figure 4A). Based on the low abun- dance of the rpL22-like short mRNA within testes, we speculated that a putative short protein product may be equally rare. To determine if the short mRNA might be translated, we analysed RNA isolated from polysomes (from male extracts) by RT-PCR (Figure 9A). rpL22-like short (and rpL22-like) mRNA was detected in polysomes (Figure 9A), suggesting that the short mRNA is translated. Based on our qRT-PCR results that indicated a large quantitative imbalance in the two mRNA isoform levels in the testis with rpL22-like short being 9800-fold less abundant (Figure 3), we increased signiﬁcantly (by nearly an order of magnitude) the amount of protein loaded for western analysis (100–120 mg compared with 15 mg) in an attempt to detect a protein that putatively might be RpL22-like short. With this protein loading strategy, additional immunodetection is seen at 25 and 13 kDa (Figure 9B and C). Notably, recombinant RpL22-like short migrates at the identical position with the 13 kDa reference band. The 25 kDa (and 13 kDa) protein may be a degradation product, only visualized with protein overloading or alternatively, the 25 kDa protein may be a post-translationally modiﬁed outcome of rpL22-like short expression. Whether or not these