In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.
In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human beta-globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and beta-globin sequences, we show that the NS1 5' splice site was effectively utilized by the beta-globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the beta-globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from beta-globin. With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.
Plotch
S J
SJ
Krug
R M
RM
D004274:DNA, Recombinant; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D009975:Orthomyxoviridae / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*
D004274:DNA, Recombinant; D012333:RNA, Messenger; D012367:RNA, Viral; D005914:Globins