A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H.
An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3'-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 approximately 60 nucleotides downstream of 3'-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5' extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3'-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two U-to-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3'-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF(35) for binding to the exon sequence flanking 3'-splice site A3. This binding results in the inhibition of splicing at 3'-splice site A3.
Jacquenet
S
S
Laboratoire de Maturation des Acide Ribo-Nucléotidique et Enzymologie Moléculaire, Unité Mixte de Recherche 7567 Université Henri Poincarré-CNRS, Boulevard des Aiguillettes, BP239, 54506 Vandoeuvre-lès-Nancy cedex, France.
Méreau
A
A
Bilodeau
P S
PS
Damier
L
L
Stoltzfus
C M
CM
Branlant
C
C
D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D005091:Exons; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C439768:hnRNP A2; D054322:tat Gene Products, Human Immunodeficiency Virus