The hnRNPs F and H2 bind to similar sequences to influence gene expression.
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 G1-5 or G12-14 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 G1-5 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, down-regulation 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.
Alkan
Serkan A
SA
Program in Biochemistry and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Martincic
Kathleen
K
Milcarek
Christine
C
D001483:Base Sequence; D015536:Down-Regulation; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation / Q000235:genetics; D009843:Oligoribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D009843:Oligoribonucleotides