A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding.
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.
Marchand
Virginie
V
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.
Méreau
Agnès
A
Jacquenet
Sandrine
S
Thomas
Denise
D
Mougin
Annie
A
Gattoni
Renata
R
Stévenin
James
J
Branlant
Christiane
C
D017398:Alternative Splicing; D001483:Base Sequence; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D014158:Transcription, Genetic; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D015694:Gene Products, rev; D015696:Gene Products, tat; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D012333:RNA, Messenger; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus