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Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1.
J Mol Biol. 1999 Feb 05; 285(5):1951-64.JM

Abstract

The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm.

Authors+Show Affiliations

Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

9925777

Citation

Najera, I, et al. "Synergistic Stimulation of HIV-1 Rev-dependent Export of Unspliced mRNA to the Cytoplasm By hnRNP A1." Journal of Molecular Biology, vol. 285, no. 5, 1999, pp. 1951-64.
Najera I, Krieg M, Karn J. Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1. J Mol Biol. 1999;285(5):1951-64.
Najera, I., Krieg, M., & Karn, J. (1999). Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1. Journal of Molecular Biology, 285(5), 1951-64.
Najera I, Krieg M, Karn J. Synergistic Stimulation of HIV-1 Rev-dependent Export of Unspliced mRNA to the Cytoplasm By hnRNP A1. J Mol Biol. 1999 Feb 5;285(5):1951-64. PubMed PMID: 9925777.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1. AU - Najera,I, AU - Krieg,M, AU - Karn,J, PY - 1999/2/2/pubmed PY - 1999/2/2/medline PY - 1999/2/2/entrez SP - 1951 EP - 64 JF - Journal of molecular biology JO - J Mol Biol VL - 285 IS - 5 N2 - The structural and accessory proteins of human immunodeficiency virus type 1 are expressed by unspliced or partially spliced mRNAs. Efficient transport of these mRNAs from the nucleus requires the binding of the viral nuclear transport protein Rev to an RNA stem-loop structure called the RRE (Rev response element). However, the RRE does not permit Rev to stimulate the export of unspliced mRNAs from the efficiently spliced beta-globin gene in the absence of additional cis-acting RNA regulatory signals. The p17gag gene instability (INS) element contains RNA elements that can complement Rev activity. In the presence of the INS element and the RRE, Rev permits up to 30 % of the total beta-globin mRNA to be exported to the cytoplasm as unspliced mRNA. Here, we show that a minimal sequence of 30 nt derived from the 5' end of the p17 gag gene INS element (5' INS) is functional and permits the export to the cytoplasm of 14% of the total beta-globin mRNA as unspliced pre-mRNA. Gel mobility shift assays and UV cross-linking experiments have shown that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and a cellular RNA-binding protein of 50 kDa form a complex on the 5' INS. Mutants in the 5' INS that prevent hnRNP A1 and 50 kDa protein binding are inactive in the transport assay. To confirm that the hnRNP A1 complex is responsible for INS activity, a synthetic high-affinity binding site for hnRNP A1 was also analysed. When the high affinity hnRNP A1 binding site was inserted into the beta-globin reporter, Rev was able to increase the cytoplasmic levels of unspliced mRNAs to 14%. In contrast, the mutant hnRNP A1 binding site, or binding sites for hnRNP C and L are unable to stimulate Rev-mediated RNA transport. We conclude that hnRNP A1 is able to direct unspliced globin pre-mRNA into a nuclear compartment where it is recognised by Rev and then transported to the cytoplasm. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/9925777/Synergistic_stimulation_of_HIV_1_rev_dependent_export_of_unspliced_mRNA_to_the_cytoplasm_by_hnRNP_A1_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(98)92473-1 DB - PRIME DP - Unbound Medicine ER -