期刊
BIOCHEMICAL JOURNAL
卷 393, 期 -, 页码 361-371出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20050538
关键词
electrophoretic mobility-shift assay (EMSA); heterogeneous nuclear ribonucleoprotein (hnRNP); polyadenylation; RNA interference (RNAi); UV-cross-linking
资金
- NATIONAL CANCER INSTITUTE [R01CA086433] Funding Source: NIH RePORTER
- NCI NIH HHS [CA86433, R01 CA086433] Funding Source: Medline
The hnRNPs (heterogeneous nuclear ribonucleoproteins) F and H2 share a similar protein structure. Both have been implicated as regulating polyadenylation, but hnRNP H2 had a positive effect, whereas hnRNP F acted negatively. We therefore carried out side-by-side comparisons of their RNA-binding and in vivo actions. The binding of the CstF2 (64 kDa cleavage stimulatory factor) to SV40 (simian virus 40) late pre-mRNA substrates containing a downstream GRS (guanine-rich sequence) was reduced by hnRNP F, but not by hnRNP H2, in a UV-cross-linking assay. Point mutations of the 14-nt GRS influenced the binding Of purified hnRNP F or H2 in parallel. Co-operative binding of the individual proteins to RNA was lost with mutations of the GRS in the G(1-5) or G(12-14) regions; both regions seem to be necessary for optimal interactions. Using a reporter green fluorescent protein assay with the GRS inserted downstream of the poly(A) (polyadenine) signal, expression in vivo was diminished by a mutant G(1-5) sequence which decreased binding of both hnRNPs (SAA20) and was enhanced by a 12-14-nt mutant that showed enhanced hnRNP F or H2 binding (SAA10). Using small interfering RNA, downregulation of hnRNP H2 levels diminished reporter expression, confirming that hnRNP H2 confers a positive influence; in contrast, decreasing hnRNP F levels had a negligible influence on reporter expression with the intact GRS. A pronounced diminution in reporter expression was seen with the SAA20 mutant for both. Thus the relative levels of hnRNP F and H2 in cells, as well as the target sequences in the downstream GRS on pre-mRNA, influence gene expression.
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