Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

Alonso-Caplen, Firelli V.; Krug, Robert M.

doi:10.1128/mcb.11.2.1092

Cited by 43 publications

(28 citation statements)

References 28 publications

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“…One hypothesis is that the 775-to-860 region in the 3' exon acts as a spacer to allow adjacent regions of NS1 mRNA to interact to form a sec- In influenza virus-infected cells, the extent of splicing of NS1 mRNA is regulated such that the steady-state amount of spliced NS2 mRNA is about 10% of that of unspliced NS1 mRNA (20,23). This regulation most likely results from a suppression in the rate of splicing coupled with the efficient transport of unspliced NS1 mRNA from the nucleus (1, 2,27,30). Recent experiments have shown that the rate of splicing of NS1 mRNA in infected cells is almost certainly controlled solely by cis-acting sequences in NS1 mRNA itself and is independent of trans-acting factors (2).…”

Section: Discussionmentioning

confidence: 99%

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.

Nemeroff¹,

Utans²,

Krämer³

et al. 1992

Mol. Cell. Biol.

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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...

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Section: Discussionmentioning

confidence: 99%

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.

Nemeroff¹,

Utans²,

Krämer³

et al. 1992

Mol. Cell. Biol.

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“…The fact that the influenza virus M1 and NS1 mRNAs are spliced by the cellular splicing machinery indicates that splicing can be uncoupled from transcription by the cellular RNA polymerase II+ However, the splicing of these two single intron viral mRNAs is inefficient: The steady-state amount of the spliced mRNA products is only about 10% of that of the unspliced mRNA precursors (Lamb et al+, 1980(Lamb et al+, , 1981+ Incomplete splicing is necessary because both the spliced and unspliced mRNAs are exported to the cytoplasm and are translated into virus-specific proteins (reviewed in Krug et al+, 1989)+ The results presented here provide an explanation for the inefficient splicing of these two single-intron influenza viral mRNAs: this splicing is not coupled to CPSF-dependent 39-end formation+ In addition, splicing which is uncoupled from polymerase II transcription may be intrinsically inefficient+ Finally, because the export of the unspliced NS1 and M1 mRNAs in influenza virus-infected cells is rapid (Alonso-Caplen & Krug, 1991), some mechanism may efficiently remove these viral mRNAs from the cellular splicing machinery+…”

Section: Implications For the Splicing Of Viral Mrnas In Influenza VImentioning

confidence: 99%

The 3′-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo

Chen

Wang³

et al. 2001

RNA

117

112

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We describe a new approach to elucidate the role of 39-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 39-end-processing machinery, is sufficient for the inhibition of not only 39-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 39-end processing, thereby establishing that 39-end processing is required for the splicing of a 39 terminal intron in vivo. Because the NS1A protein causes a global suppression of 39-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 39-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 39-end formation. In contrast, the 39-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.

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“…This suggests that at least for these viruses, the splicing of NS1 mRNA depends solely on a cis-acting sequence in the NS1 mRNA itself, consistent with the results obtained from in vitro splicing of NS1 mRNA (Nemeroff et al, 1992) and the NS2 protein functioning from the early phase of infection. The splicing of NS1 mRNA has also been shown to depend on the efficiency of nucleocytoplasmic transport of the unspliced NS1 mRNA (Alonso-Caplen & Krug, 1991).…”

Section: Introductionmentioning

confidence: 99%

An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis

Odagiri¹,

Tominaga

Tobita³

et al. 1994

Journal of General Virology

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The mutated non-structural NS2 protein of an influenza A virus mutant, Wa-182, has been shown to be responsible for the production of defective interfering (DI) particles lacking the PA gene after a single cycle high-multiplicity infection. Using a subclone of Wa-182, A3/e-3, that inherited the Wa-182 phenotype but contained only a marginal amount of DI RNAs derived from the PA gene, we showed that replication of the PA genome RNA was suppressed primarily at the step of complementary RNA (cRNA) synthesis. On the other hand, the small amounts of DI RNA species present in the stock of A3/e-3 were shown to be replicated efficiently. These findings suggested that the suppression of cRNA synthesis of the PA gene was caused by preferential amplification of the DI RNAs. The suppression of PA gene cRNA synthesis subsequently resulted in suppression of both virion RNA synthesis and secondary transcription of the PA gene. Such aberrant replication of the PA gene was found to be attributable to an amino acid change in the NS2 protein at position 32, from isoleucine to threonine. These results suggest that the NS2 protein plays a role in promoting normal replication of the genomic RNAs by preventing the replication of short-length RNA species.

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Regulation of the extent of splicing of influenza virus NS1 mRNA: role of the rates of splicing and of the nucleocytoplasmic transport of NS1 mRNA.

Cited by 43 publications

References 28 publications

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.

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.

The 3′-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo

An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis

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