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. 2010 Apr 27;8(4):e1000360. doi: 10.1371/journal.pbio.1000360

Figure 6. Mex67p is a component of the cytoplasmic YRA1 pre-mRNP that functions in repressing YRA1 pre-mRNA translation.

Figure 6

(A) Analysis of the effect of inactivation of Mex67p on the translation of YRA1 pre-mRNA. upf1Δedc3Δmex67-5 cells were grown at 25°C and then shifted to 37°C for 6 min. The polyribosomal association of YRA1 pre-mRNA and mRNA in these cells before or after the temperature shift was analyzed by sucrose gradient fractionation and Northern blotting. Upper panels: absorbance tracings at 254 nm; lower panels: Northern blots of individual gradient fractions. Blots were hybridized with a probe complementary to YRA1 transcripts. The percentages of YRA1 pre-mRNA and mRNA in the mRNP and the polyribosomal fractions are indicated. (B) Analysis of the association of Mex67p with YRA1 pre-mRNA. Whole cell extracts from upf1Δ edc3Δ strains harboring either the MEX67 or the HA-MEX67 allele were incubated with anti-HA antibodies. Proteins and RNAs precipitated by the antibodies were analyzed by Western blotting (left panel) and RT-PCR (right panel). I, input; S, supernatant fraction; P, pellet fraction. HA-Mex67p and specific RT-PCR products for YRA1 and CYH2 pre-mRNAs were detected in the pellet fraction. RT, reverse transcriptase. (C) Analysis of the effect of tethering Mex67p on YRA1 pre-mRNA decay. A DNA fragment containing two MS2-coat protein binding sites was inserted into the intronic region of the F7, R1-F7, F12, and N-400 alleles of YRA1. The steady-state levels of the YRA1 pre-mRNA transcripts encoded by the resulting F7-MS2, R1-F7-MS2, F12-MS2, and N-400-MS2 alleles in wild-type (1), upf1Δ (2), edc3Δ (3), and upf1Δedc3Δ (4) cells that do or do not express the MS2-coat- Mex67p or Sub2p fusion proteins were determined by Northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of the endogenous and exogenous YRA1 pre-mRNAs and YRA1 mRNA are indicated. A schematic diagram of the analyzed alleles is shown above the Northern blot, with the relative positions of the MS2-binding sites, the intron modules, and the intron deletions indicated.