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A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding.
J Mol Biol. 2002 Nov 01; 323(4):629-52.JM

Abstract

Retroviral protein production depends upon alternative splicing of the viral transcript. The HIV-1 acceptor site A7 is required for tat and rev mRNA production. Production of the Tat transcriptional activator is highly controlled because of its apoptotic properties. Two silencer elements (ESS3 and ISS) and two enhancer elements (ESE2 and ESE3/(GAA)3) were previously identified at site A7. hnRNP A1 binds ISS and ESS3 and is involved in the inhibitory process, ASF/SF2 activates site A7 utilisation. Here, by using chemical and enzymatic probes we established the 2D structure of the HIV-1(BRU) RNA region containing site A7 and identified the RNA segments protected in nuclear extract and by purified hnRNP A1. ISS, ESE3/(GAA)3 and ESS3 are located in three distinct stem-loop structures (SLS1, 2 and 3). As expected, hnRNP A1 binds sites 1, 2 and 3 of ISS and ESS3b, and oligomerises on the polypurine sequence upstream of ESS3b. In addition, we discovered an unidentified hnRNP A1 binding site (AUAGAA), that overlaps ESE3/(GAA)3. On the basis of competition experiments, hnRNP A1 has a stronger affinity for this site than for ESS3b. By insertion of (GAA)3 alone or preceded by the AUA trinucleotide in a foreign context, the AUAGAA sequence was found to modulate strongly the (GAA)3 splicing enhancer activity. Cross-linking experiments on these heterologous RNAs and the SLS2-SLS3 HIV-1 RNA region, in nuclear extract and with recombinant proteins, showed that binding of hnRNP A1 to AUA(GAA)3 strongly competes the association of ASF/SF2 with (GAA)3. In addition, disruption of AUA(GAA)3 demonstrated a key role of this sequence in hnRNP A1 cooperative binding to the ISS and ESS3b inhibitors and hnRNP A1 oligomerisation on the polypurine sequence. Thus, depending on the cellular context ([ASF/SF2]/[hnRNP A1] ratio), AUA(GAA)3 will activate or repress site A7 utilisation and can thus be considered as a Janus splicing regulator.

Authors+Show Affiliations

Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567 UHP-CNRS, Université Henri Poincaré Nancy 1, Boulevard des Aiguillettes, BP239, 54506 Cedex, Vandoeuvre-lès-Nancy, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12419255

Citation

Marchand, Virginie, et al. "A Janus Splicing Regulatory Element Modulates HIV-1 Tat and Rev mRNA Production By Coordination of hnRNP A1 Cooperative Binding." Journal of Molecular Biology, vol. 323, no. 4, 2002, pp. 629-52.
Marchand V, Méreau A, Jacquenet S, et al. A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J Mol Biol. 2002;323(4):629-52.
Marchand, V., Méreau, A., Jacquenet, S., Thomas, D., Mougin, A., Gattoni, R., Stévenin, J., & Branlant, C. (2002). A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. Journal of Molecular Biology, 323(4), 629-52.
Marchand V, et al. A Janus Splicing Regulatory Element Modulates HIV-1 Tat and Rev mRNA Production By Coordination of hnRNP A1 Cooperative Binding. J Mol Biol. 2002 Nov 1;323(4):629-52. PubMed PMID: 12419255.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. AU - Marchand,Virginie, AU - Méreau,Agnès, AU - Jacquenet,Sandrine, AU - Thomas,Denise, AU - Mougin,Annie, AU - Gattoni,Renata, AU - Stévenin,James, AU - Branlant,Christiane, PY - 2002/11/7/pubmed PY - 2002/12/6/medline PY - 2002/11/7/entrez SP - 629 EP - 52 JF - Journal of molecular biology JO - J Mol Biol VL - 323 IS - 4 N2 - Retroviral protein production depends upon alternative splicing of the viral transcript. The HIV-1 acceptor site A7 is required for tat and rev mRNA production. Production of the Tat transcriptional activator is highly controlled because of its apoptotic properties. Two silencer elements (ESS3 and ISS) and two enhancer elements (ESE2 and ESE3/(GAA)3) were previously identified at site A7. hnRNP A1 binds ISS and ESS3 and is involved in the inhibitory process, ASF/SF2 activates site A7 utilisation. Here, by using chemical and enzymatic probes we established the 2D structure of the HIV-1(BRU) RNA region containing site A7 and identified the RNA segments protected in nuclear extract and by purified hnRNP A1. ISS, ESE3/(GAA)3 and ESS3 are located in three distinct stem-loop structures (SLS1, 2 and 3). As expected, hnRNP A1 binds sites 1, 2 and 3 of ISS and ESS3b, and oligomerises on the polypurine sequence upstream of ESS3b. In addition, we discovered an unidentified hnRNP A1 binding site (AUAGAA), that overlaps ESE3/(GAA)3. On the basis of competition experiments, hnRNP A1 has a stronger affinity for this site than for ESS3b. By insertion of (GAA)3 alone or preceded by the AUA trinucleotide in a foreign context, the AUAGAA sequence was found to modulate strongly the (GAA)3 splicing enhancer activity. Cross-linking experiments on these heterologous RNAs and the SLS2-SLS3 HIV-1 RNA region, in nuclear extract and with recombinant proteins, showed that binding of hnRNP A1 to AUA(GAA)3 strongly competes the association of ASF/SF2 with (GAA)3. In addition, disruption of AUA(GAA)3 demonstrated a key role of this sequence in hnRNP A1 cooperative binding to the ISS and ESS3b inhibitors and hnRNP A1 oligomerisation on the polypurine sequence. Thus, depending on the cellular context ([ASF/SF2]/[hnRNP A1] ratio), AUA(GAA)3 will activate or repress site A7 utilisation and can thus be considered as a Janus splicing regulator. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/12419255/A_Janus_splicing_regulatory_element_modulates_HIV_1_tat_and_rev_mRNA_production_by_coordination_of_hnRNP_A1_cooperative_binding_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022283602009671 DB - PRIME DP - Unbound Medicine ER -