RanBP2/Nup358 Potentiates the Translation of a Subset of mRNAs Encoding Secretory Proteins (original) (raw)
Figure 1
ALREX-promoting SSCRs promote translation.
(A) For each codon in the MHC, 2Ile-MHC SSCRs (_x_-axis), the frequency with which it appears in the human genome per 1,000 codons (“Freq/1000”; _y_-axis) was plotted. Note that the two leucine-to-isoleucine mutations are circled. (B) Codon analysis of Ins, 2Ile-Ins SSCRs as in (A). (C–L) U2OS cells were co-transfected with plasmids that contained various versions of the ftz gene, and a second plasmid that either contained (“+”) or lacked (“−”) the H1B-GFP gene. As a control, cells were transfected with a plasmid lacking the ftz gene (“Vector”). The cells were then analyzed 18–24 h post-transfection. (C–D) Cell lysates were separated by SDS-PAGE and were probed with antibodies against the HA epitope, GFP, and α-tubulin. (E–F) RNA was extracted from the cell lysates, separated on a denaturing agarose gel, and analyzed by Northern blotting using [32P]-labeled probes directed against ftz and GFP transcripts. (G–H) The percentage of total mRNA found in the cytoplasm and nucleus as determined by the distribution of ftz mRNA by FISH staining. Each bar represents the average and standard error between three separate experiments (each experiment consisting of the average of at least 30 cells). (I–L) For cells expressing the indicated version of ftz, the percentage of cells (_y_-axis) with various numbers of SGs (_x_-axis) was plotted. Note that the SGs were detected by Tia1 immunostaining, but almost always contained an enrichment in ftz mRNA. For each graph, >160 cells (I–J) and >580 cells (K–L) were analyzed. Note that while >15% of 2Ile-INS-ftz expressing cells had SGs, this number dropped to <1% in cells expressing INS-ftz. (M) For the first 17 codons (_x_-axis) in the wild type and 4Ile mutant CALR gene, the frequency with which it appears in the human genome per 1,000 codons (_y_-axis). Note that the four leucine-to-isoleucine mutations in 4Ile-CALR are circled. (N–P) U2OS cells were co-transfected with plasmids that contained either version of the HA-tagged CALR gene, and a second plasmid that either contained (“+”) or lacked (“−”) the H1B-GFP gene. As a control, cells were transfected with a plasmid lacking the CALR gene (“Vector”). The cells were analyzed 18–24 h post-transfection. (N) Cell lysates were separated by SDS-PAGE and were probed with antibodies against the HA epitope, GFP, and α-tubulin. (O) RNA was extracted from the cell lysates, separated on a denaturing gel and analyzed by Northern blotting analysis using [32P]-labeled probes directed against CALR and GFP RNA. (P) The percentage of total mRNA found in the cytoplasm and nucleus as determined by the distribution of CALR mRNA by FISH staining.