A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

Agris, Cheryl H.; Nemeroff, M E; Krug, Robert M.

doi:10.1128/mcb.9.1.259

Cited by 27 publications

(34 citation statements)

References 33 publications

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“…It was shown previously that despite the almost total absence of splicing intermediates in the in vitro reaction, intact NS1 mRNA efficiently formed ATP-dependent 55S complexes containing the Ul, U2, U4, U5, and U6 snRNPs (1). If these 55S complexes represented authentic spliceosome species that were, however, blocked in the subsequent catalysis of splicing, then intact NS1 mRNA and spliceable NS1 mRNA lacking the inhibitory exon and intron sequences should form 55S complexes with similar, if not identical, efficiencies.…”

Section: Resultsmentioning

confidence: 99%

“…This type of splicing regulation also occurs with retroviruses. In in vitro splicing reactions, NS1 mRNA forms 55S complexes containing the Ul, U2, U4, U5, and U6 snRNPs, but little or no catalysis of the first step in splicing occurs (1,28). The cause of this block in catalysis was not determined, but the available data argued against the involvement of the 5' and 3' splice sites and branchpoint of NS1 mRNA in this block.…”

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confidence: 90%

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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: Resultsmentioning

confidence: 99%

mentioning

confidence: 90%

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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“…Although a similar effect was not observed with a large truncation of the env pre-mRNA intron including these elements (Katz and Skalka 1990), the negative elements may function only in a certain sequence context. Negative elements may also play a role in incomplete splicing of influenza virus NSl mRNA (Plotch and Krug 1986;Agris et al 1989).…”

Section: Discussionmentioning

confidence: 99%

The role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

Fu¹,

Katz²,

Skalka³

et al. 1991

Genes Dev.

110

128

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We previously described an avian sarcoma-leukosis virus (ASLV) insertion mutation that causes a decrease in the ratio of unspliced to spliced RNA in vivo, resulting in a replication defect. Pseudorevertant viruses containing cis-acting suppressor mutations that restored the normal ratio were isolated. One class of the suppressor mutations consists of single-base changes or small deletions near the 3' splice site, while another consists of deletions in the 3' exon. In this paper we report results from an in vitro analysis of wild-type, mutant, and pseudorevertant pre-mRNA splicing. We find that wild-type RNA is spliced inefficiently in vitro, and that the insertion mutation and suppressors act directly at the level of splicing. Characterization of splicing intermediates reveals that the insertion mutation and suppressor mutations located within the intron alter the pattern of lariat formation. In contrast, suppressor mutations consisting of 3' exon deletions act at an earlier step in the splicing pathway. Thus, the efficiency of splicing at the env 3' splice site can be affected at the level of spliceosome assembly, lariat formation, or cleavage at the 3' splice site and exon ligation.[Key Words: Retrovirus; splicing; splice site selection] Received September 18, 1990; revised version accepted December 13, 1990.Retrovirus RNA transcripts are subject to multiple pathways of RNA processing and transport (for a review, see Stoltzfus 1989). The primary full-length RNA po7lI transcript of -7-10 kb functions as genomic RNA for progeny virions and mRNA for structural proteins and enzymes. Both of these functions require transport of an intact viral RNA to the cytoplasm. In addition, a fraction of the full-length transcripts is spliced in the nucleus to generate subgenomic mRNAs that encode other viral proteins (Fig. lA). Thus, a balance between full-length RNA and spliced viral mRNAs is required for replication of retroviruses.Multiple CIS-acting sequences have been shown to play a role in splice site selection in cellular pre-mRNAs (for review, see Krainer and Maniatis 1988). These sequences include the 5' and 3' splice sites, the branchpoint sequence (BPS), and sequences within the flanking exons. Interaction of these cis-acting elements with small nuclear ribonuclear proteins (snRNPs) and many protein splicing factors leads to the assembly of spliceosomes in which the splicing reaction takes place (see Krainer and Maniatis 1988;Steitz et al. 1988; for recent reviews, see Bindereif and Green 1990).Retroviral transcripts are presumably processed by the same mechanisms as cellular pre-mRNAs. However, unlike most cellular pre-mRNAs, a portion of viral RNA transcripts escape splicing entirely. Balanced splicing of retroviral RNA is therefore a novel form of alternative splicing. An understanding of the mechanisms involved in this splicing may reveal both general and novel features of the splice site selection process. Several models for maintaining the balance of spliced and unspliced forms of retrovirus RNA have been pro...

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“…The RNA products were analyzed by gel electrophoresis. The identities of the premRNA and splicing products, which have been established previously (Fumeaux et al 1985;Perkins et al 1986;Agris et al 1989;Krainer et al 19901, are indicated schematically: [] and 9 correspond to the 5' and 3' exons, respectively; thin lines correspond to the intron, which is converted to a lariat form during splicing.…”

Section: The Ns1 Protein Inhibits Pre-mrna Splicing In Vitromentioning

confidence: 99%

“…A [3-globin minigene in pGEM1 linearized with EcoRI and the adenovirus pKT1 minigene in sp65 linearized with ScaI were transcribed with SP6 RNA polymerase, as described previously (Agris et al 1989). The in vitro transcripts contained -3x 107 cpm (derived from [a-g2P]UTP/~g and contained mZGpppG 5' ends.…”

Section: In Vitro Splicing Assays and Native Gel Electrophoresismentioning

confidence: 99%

The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.

Lu¹,

Qian²,

1994

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We have shown previously that the influenza virus NS1 protein inhibits the nuclear export of mRNAs. Here we demonstrate that the NS1 protein also regulates another post-transcriptional step: It inhibits pre-mRNA splicing both in vivo and in vitro. The mode by which the NS1 protein inhibits pre-mRNA splicing is novel. The pre-mRNA forms spliceosomes, but subsequent catalytic steps in splicing are inhibited. Affinity selection experiments establish that the NS1 protein is associated with the spliceosomes that are formed. The RNA-binding domain of the NSI protein is required for the inhibition of splicing and for the interaction of the protein with spliceosomes. Because the NS1 protein is associated with U6 snRNA in influenza virus-infected cells as well as in splicing extracts from uninfected cells, it is likely that the NS1 protein also inhibits pre-mRNA splicing in infected cells. Surprisingly, the splicing of the viral nsl mRNA, the very mRNA that encodes the NS1 protein, was resistant to inhibition by the NS1 protein. This resistance is conferred by sequences in nsl mRNA.[Key Words: Influenza virus NS1 protein; pre-mRNA splicing; spliceosome; U6 snRNA] Received May 12, 1994; revised version accepted June 16, 1994.Eukaryotic pre-mRNAs synthesized by nuclear RNA polymerase II undergo a series of post-transcriptional processes. Two of these processes are pre-mRNA splicing and mRNA export from the nucleus, both of which can be regulated (Green 1989;Krug 1993). Alternative splicing, whereby different mRNAs are generated from a single pre-mRNA, has been shown to be regulated by proteins that bind to specific regions of the pre-mRNA precursor. In many instances it has been shown that specific proteins act on specific pre-mRNA targets to regulate their alternative splicing. For example, in the Drosophila sex determination pathway, a specific protein(s)--sex lethal protein or the complex of the Tra and Tra2 proteins--binds to a specific site on a particular premRNA to either suppress or activate, respectively, the usage of a 3' splice site (Baker 1989;Inoue et al. 1990;Bell et al. 1991;Tian and Maniatis 1992).The nuclear export of unspliced pre-mRNAs and spliced mRNAs can also be regulated by specific proteins. Usually unspliced pre-mRNAs are not exported from the nucleus, apparently at least in part because the pre-mRNAs are efficiently committed to spliceosome formation in the nucleus (Legrain and Rosbash 1989). However, this process is overcome in the case of the pre-mRNAs of human immunodeficiency virus-1 (HIV-1) and other lentiviruses by the action of the virus-encoded Rev (or Rev-like) protein, which binds to a specific RNA sequence (RRE, or Rev responsive element) in the viral pre-mRNAs and facilitates their nuclear export (Hadzopoulou-Cladaras et al. 1989; Hanly et al. 1989;Malim et al. 1989). The influenza virus NS1 protein has the opposite effect on the nuclear export of mRNA: It inhibits the transport of mRNAs (Alonso-Caplen et al. 1992;Fortes et al. 1994). The NS1 protein binds to the poly(A) sequence at t...

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A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

Cited by 27 publications

References 33 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 role of branchpoint and 3'-exon sequences in the control of balanced splicing of avian retrovirus RNA.

The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing.

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