Identification of cis-acting intron and exon regions in influenza virus NS1 mRNA that inhibit splicing and cause the formation of aberrantly sedimenting presplicing complexes.

Abstract

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the Ul, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) (C. H. Agris, M. E. Nemeroff, and R. M. Krug, Mol. Cell. Biol. 9:259-267, 1989). We demonstrate that the block in splicing was caused by two regions in NS1 mRNA: (i) a large intron region (not including the branchpoint sequence) and (ii) an 85-nucleotide 3' exon region near the 3' end of the exon. After removal of both of these regions, the 5' and 3' splice sites and branchpoint of NS1 mRNA functioned efficiently in splicing, indicating that they were not defective. The two inhibitory regions shared one property: splicing inhibition was independent of the identity of the nucleotide sequence in either region. In other respects, however, the two inhibitory regions differed. The inhibitory activity of the intron region was proportional to its length, indicating that the inhibition was probably due to size only. In contrast, the 3' exon, which was of small size, was a context element; i.e., it functioned only when it was located at a specific position in the 3' exon of NS1 mRNA. To determine how these intron and exon regions inhibited splicing, we compared the types of splicing complexes formed by intact NS1 mRNA with those formed by spliceable NS1 mRNA lacking the intron and exon regions. Splicing complexes were formed by using purified splicing factors. The most definitive results were obtained under conditions in which only ATP-dependent presplicing 30S complexes were formed with spliceable NS1 mRNA: intact NS1 mRNA still formed ATP-dependent complexes sedimenting at about 55S, even though the protein components needed for the formation of authentic 55S spliceosomes were not present. We postulate that the NS1 mRNA intron and exon regions inhibit splicing by not allowing NS1 mRNA to be folded properly into a substrate for splicing. In fact, the small 85-nucleotide-long 3' exon region by itself may block proper folding of NS1 mRNA, as the ATP-dependent presplicing complexes formed with NS1 mRNA lacking the intron region also sedimented aberrantly, at approximately 45S rather than 30S.The possible role of the regions in the regulation of NS1 mRNA splicing in vivo is discussed.During the initial phase of in vitro splicing, pre-mRNA substrates are assembled into large 5OS-60S complexes containing five small nuclear ribonucleoproteins (snRNPs), the Ul, U2, U4, U5, and U6 snRNPs (4,6,9,12,26). The Ul and U2 snRNPs bind to the 5' splice site and intron branchpoint of the pre-mRNA, respectively (5, 24, 34); the U5 snRNP probably binds to the 3' splice site (7,25). These large complexes, or spliceosomes, contain the products of the first step in splicing, the 5' exon and lariat-3' exon (4, 6, 9, 12, 26).Influenza virus NS1 mRNA is spliced to form a smaller mRNA, NS2 mRNA. Because both the unspliced (NS1) and spliced (NS2) mRNAs code for proteins, the extent of splicing is regulated in infected...

Identification of potential conserved RNA secondary structure throughout influenza A coding regions

“…Others have noted that RNA secondary structure may play an important role in splicing of influenza virus segment 8 (Plotch and Krug 1986;Nemeroff et al 1992). Interestingly, segment 7 RNA displays a similar pattern of predicted secondary structure surrounding the 59 and 39 splice sites (Figs.…”

“…Segment 8 (+)RNA has been the most extensively studied with regard to secondary structure. RNA secondary and tertiary structures have also been proposed to be important in the splicing of segment 8 mRNA (Plotch and Krug 1986;Nemeroff et al 1992) and in viral packaging of (À)RNA (Muramoto et al 2006;Marsh et al 2007Marsh et al , 2008Hutchinson et al 2008;Liang et al 2008). Outside of segment 8, very little is known about RNA secondary structure.…”

Influenza virus NS1 protein alters the subnuclear localization of cellular splicing components

“…However, these splicing events are regulated to permit the concurrent expression of the NS1 and M1 proteins, which are produced from the collinear mRNAs and are also essential for the virus infection to proceed. Both cis-and trans-acfing elements appear to mediate the control of these processes (Nemeroff et al, 1992;Smith & Inglis, 1985;Valcfircel et al, 1991) and the NS1 protein itself might be responsible for such regulation, * Author for correspondence. Fax 34 1 585 4506. e-mail JORTIN @SAMBA.CNB.UAM.ES since it alters the splicing pattern of NS1 mRNA and other cellular mRNAs (Fortes et al, 1994).…”

A single polypyrimidine tract binding protein (PTB) binding site mediates splicing inhibition at mouse IgM exons M1 and M2

“…The splicing inhibitors identified to date appear remarkably diverse (Nemeroff et al 1992;Siebel et al 1992;Amendt et al 1995;Del Gatto and Breathnach 1995;Staffa and Cochrane 1995;Staffa et al 1997;Valcarcel and Gebauer 1997;Grabowski 1998;Zheng et al 1998;Kan and Green 1999;Chou et al 2000;Le Guiner et al 2001;Wagner and Garcia-Blanco 2001). Several proteins with the ability to inhibit splicing have been identified, including two heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1 and polypyrimidine tract binding protein (PTB or hnRNP I).…”