2712 Nucleic Acids Research, 2011, Vol. 39, No. 7 Figure 9. rpL22-like short mRNA may be translated. (A) Whole male extract was subjected to 10–50% linear sucrose gradient ultra-centrifugation. RT-PCR analysis of gradient fractions representing 80S subunits and polysomes shows association of rpL22-like (arrow) and rpL22-like short (arrowhead) mRNAs with active translating ribosomes, suggesting rpL22-like short is translated. (B) An initial western analysis of 75 mg of testis extract shows immunodetection at the expected MW of 34 kDa and lower at 25 and 13 kDa (faint band; arrow). ‘Short exposure’ was for 3 s and ‘long exposure’ was for 30 min. (C) Increased protein loading to 120 mg of testis extract enhanced immunodetection of the 13 kDa band (arrow) with a longer exposure (30 min compared to 4 s for short exposure). Given the abundance of RpL22-like as a ‘sink’ for antiRpL22-like Ab, the membrane was cut to maximize immunodetection of smaller MW proteins (arrowhead). The 13 kDa band (arrow) aligns with the recombinant RpL22-like short protein standard. M: pGEM marker; NTC: no template control; E: male extract; B: bottom of gradient; RS: recombinant standard. genes. In this study, RpL22-like escaped identiﬁcation as a paralogue and as an up-regulated gene in testes because its homology to RpL22 fell below the minimum 50% homology threshold. Microarray data from FlyAtlas suggest that the levels of rpL22 and rpL22-like mRNAs in testis are comparable. RpL22-like may augment a func- tion(s) of RpL22 by providing a testis-speciﬁc ribosomal role under speciﬁc developmental or physiological conditions. Outside of the reproductive system, RpL22-like is found within head tissue in possibly the eye (but in lower abun- dance compared with testes) from both sexes in a struc- tural conformation that is distinct from the testis conformation, suggesting a possible alternative functional role for RpL22-like in the head. Although rpL22-like mRNA levels in male and female heads are low based on qRT-PCR results (Figure 3), higher levels of rpL22-like expression in the relevant tissue in the head may be masked by the abundance of other tissue types. Interestingly, the quantity of rpL22-like mRNA measured in heads and ovaries is similar (Figure 3); yet, protein ex- pression differs considerably with no protein detected in ovaries. We have conﬁrmed the prediction that RpL22-like is a ribosomal component based on its co-sedimentation with gradient-puriﬁed ribosomes and polysomes from male extracts. Taken together with quantitative expression data from testis, we infer that RpL22-like is a component of testis ribosomes. The majority of RpL22-like in male extracts was found in association with ribosomes or poly- somes (and little, if any, was detected at the top of gradient proﬁles), suggesting that RpL22-like is a more permanent ribosomal component than has been determined for RACK1 [see review by ref. (3)]. Yet, not necessarily a protein with an exclusively ribosomal function, RpL22-like may have an alternate role(s) as well. Though it is known that RpL22 is ubiquitously ex- pressed (e.g. shown in the entire male reproductive tract by IHC), it is unclear if RpL22 functions exclusively as an RP within the testis. Differences in RpL22-like structure compared with its paralogue RpL22 are most apparent in the N-terminal domain; however, there are also C-terminal amino acid differences that may contribute to functional differences between RpL22-like and RpL22. A small C-terminal ex- tension of nine amino acid is apparent at the very C-terminal end of RpL22-like. The recently published 5.5 A˚ model of the eukaryotic 80S ribosome shows the position of RpL22 on the 60S subunit surface near its base (12). In this model, the N- and C-terminal segments of RpL22 are positioned near each other, extending from the subunit surface, which may allow for interactions with other components. If paralogue binding is mutually exclu- sive (though our data do not discount a model in which