Nucleic Acids Research, 2011, Vol. 39, No. 7 2709 Figure 6. Density gradient ultra-centrifugation showing L22-like association with active translational machinery. Soluble extracts from adult male ﬂies were fractionated on 10–50% sucrose gradients. The top left panel shows the absorbance proﬁle of the sucrose gradient. Extracted RNAs from fractions are shown on the top right. Fractions containing polysomes (poly) and 80S ribosomes were pooled, pelleted separately and subjected to western analysis (bottom panels). Both paralogues were detected at the expected MW in fractions containing 80S ribosomes and polysomes, indicating that both are stable components of translating ribosomes. No sizeable amount of either protein was detected at the top (T) of the gradient. Input: 25 mg and/or 50 mg whole male extract. product sizes: 950 bp for rpL22-like and 392 bp for rpL22-like short using an exon1/2 bridge primer, and 370 bp derived from rpL22-like short using the novel exon2/3 bridge primer (Figure 8A). Additional control RT-PCR experiments to rule out artifactual ampliﬁcation of a shortened transcript were performed using in vitro synthesized full-length and short transcripts derived from cloned cDNA tem- plates and ﬂanking or bridge primer sets (Supplementary Figure S1). With ﬂanking primers and full-length in vitro synthesized RNA, a single amplicon of 939 bp was generated, as expected. Notably low MW amplicons rep- resenting rpL22-like short cDNAs were not produced. Using in vitro short RNA as a template, an expected amplicon of 392 bp was generated. Only when bridge primer sets (novel exon2/exon3) were used with short in vitro RNA templates were ampliﬁed products of the expected size generated; no amplicons were produced with bridge primers and full-length in vitro RNA. We conclude that the low MW amplicon generated from in vivo polyA+ mRNA templates from different develop- mental stages as well as from various tissues is an rpL22- like mRNA variant and not an artifact of aberrant RT-PCR ampliﬁcation. Further evidence for the presence of lower MW rpL22- like mRNA isoforms was shown in northern blot analyses using embryonic, larval and/or adult polyA+ RNA and 50 and 30 ﬂanking rpL22-like oligonucleotide probes that would detect both full-length and short mRNAs or an intron-speciﬁc probe that would detect full-length mRNA and the ‘retained’ intron (if sufﬁciently stable). It should be noted that the bridge primer used in RT-PCR experiments described above would not be expected to detect rpL22-like short mRNA exclusively since the majority of sequences in that probe would also hybridize to full-length mRNA under the standard hybrid- ization conditions used. Using probes that should detect both full- and short- mRNAs, we identiﬁed in each developmental stage, two prominent hybridization signals at 0.7 and 0.5 kb (in addition to a signal at 1.2 kb) that are smaller in size than the length expected for full-length rpL22-like mRNA. The 0.7 kb species falls within the range of the minimum size for an rpL22-like short transcript at 0.625 kb, not accounting for possible variation in polyadenylation (Figure 8B). The lower MW RNA species are less abundant than the RNA species at 1.2 kb (which likely represents full-length rpL22-like mRNA)—an expected quantitative result for rpL22-like short mRNA based on qRT-PCR results (Figure 3). The identity of the 0.5 kb species is unknown; however, it is unlikely to represent the excised intron itself since ﬂanking rpL22-like probes would not hybridize exclusively to retained intron sequences. Interestingly, in northern blot experiments where embryo polyA+ RNA (different sample than used in Figure 8B) was initially probed with an intron-speciﬁc probe, we detected an RNA of the size expected for full- length rpL22-like mRNA and a lower MW species that may represent the excised intron of 0.57 kb (Figure 8C). The putative intron species is less abundant than the full- length species (expected based on the relative amount of spliced variant compared to full-length mRNA from qRT-PCR data; Figure 3) and would only be detected as