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

In in vitro splicing reactions, influenza virus NS1 mRNA was not detectably spliced, but nonetheless very efficiently formed ATP-dependent 55S complexes containing the U1, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components.

RNA ◽

10.1261/rna.078329.120 ◽

2021 ◽

pp. rna.078329.120

Author(s):

Valentin Jacquier ◽

Manon Prevot ◽

Thierry Gostan ◽

Remy Bordonne ◽

Sofia Benkhelifa-Ziyyat ◽

...

Keyword(s):

Muscular Atrophy ◽

Differential Splicing ◽

Molecular Determinants ◽

In Vitro Splicing ◽

Splicing Efficiency ◽

Smn Protein ◽

Survival Of Motor Neuron ◽

Branchpoint Sequence ◽

First Time

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

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The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

Biochemical Journal ◽

10.1042/bj20081195 ◽

2008 ◽

Vol 416 (3) ◽

pp. 365-374 ◽

Cited By ~ 9

Author(s):

Aparna K. Sapra ◽

Piyush Khandelia ◽

Usha Vijayraghavan

Keyword(s):

Second Step ◽

Splicing Factors ◽

Temperature Sensitive ◽

Biochemical Basis ◽

Association Analyses ◽

Small Nuclear Rnas ◽

In Vitro Splicing ◽

Mechanistic Basis ◽

Small Nuclear Ribonucleoproteins

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 °C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Δ extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17–spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Δ and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.

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Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5273 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5273-5280 ◽

Cited By ~ 2

Author(s):

G C Conway ◽

A R Krainer ◽

D L Spector ◽

R J Roberts

Keyword(s):

Rna Splicing ◽

De Novo ◽

Mrna Splicing ◽

Splicing Factors ◽

Macromolecular Complex ◽

Large Structures ◽

Nuclear Extracts ◽

In Vitro Splicing ◽

Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

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Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

eLife ◽

10.7554/elife.27402 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Andrea Pawellek ◽

Ursula Ryder ◽

Triin Tammsalu ◽

Lewis J King ◽

Helmi Kreinin ◽

...

Keyword(s):

Complex Formation ◽

Splicing Factors ◽

Nuclear Speckles ◽

E Coli ◽

Sumo Protease ◽

Complex Cells ◽

U2 Snrnp ◽

In Vitro Splicing ◽

In Cells

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, E. coli expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.

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The 216-nucleotide intron of the E1A pre-mRNA contains a hairpin structure that permits utilization of unusually distant branch acceptors.

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4852 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4852-4861 ◽

Cited By ~ 35

Author(s):

K Chebli ◽

R Gattoni ◽

P Schmitt ◽

G Hildwein ◽

J Stevenin

Keyword(s):

Splice Site ◽

Site Directed Mutagenesis ◽

Hairpin Structure ◽

Cis Acting ◽

Adenovirus E1a ◽

Sequence Elements ◽

In Vitro Splicing ◽

Hairpin Formation

A recently characterized 216-nucleotide intron-splicing reaction occurs within the adenovirus E1A pre-mRNA through the use of three branch acceptor sites, located at 59, 55, and 51 nucleotides from the 3' splice site. To investigate the role of the cis-acting sequence elements in the selection of such unusually distant branch sites, transcripts differing in sequence downstream of the branch sites were analyzed for in vitro splicing. Initial results suggested that secondary structure could be involved in the use of distant branch sites. The involvement of a hairpin structure, including a nine-G C-base-pair stem, was supported by the results of site-directed mutagenesis analyses. Mutations that destroyed or weakened this hairpin resulted in an inefficient splicing reaction. In contrast, complementary mutation or deletion of two bulges, which involved a restoration or reinforcement of the hairpin, resulted in a reactivation or improvement of the splicing efficiency, respectively. Therefore, we conclude that the hairpin structure shortens the operational distance between the 3' splice site and the branch acceptors and brings the branch sites into the branch-permissive window, 18 to 40 nucleotides upstream of the 3' splice site. Our results confirm the importance of the constraint of distance for the splicing reaction and show that this constraint may be overcome by means of a stable hairpin formation.

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Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.74.13.5902-5910.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5902-5910 ◽

Cited By ~ 13

Author(s):

Zhi-Ming Zheng ◽

Jesse Quintero ◽

Eric S. Reid ◽

Christian Gocke ◽

Carl C. Baker

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Splicing Factors ◽

Bovine Papillomavirus ◽

Splicing Pattern ◽

In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

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Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing

Molecular and Cellular Biology ◽

10.1128/mcb.9.12.5273-5280.1989 ◽

1989 ◽

Vol 9 (12) ◽

pp. 5273-5280

Author(s):

G C Conway ◽

A R Krainer ◽

D L Spector ◽

R J Roberts

Keyword(s):

Rna Splicing ◽

De Novo ◽

Mrna Splicing ◽

Splicing Factors ◽

Macromolecular Complex ◽

Large Structures ◽

Nuclear Extracts ◽

In Vitro Splicing ◽

Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

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Participation of the C-Terminal Domain of RNA Polymerase II in Exon Definition during Pre-mRNA Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8290-8301.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8290-8301 ◽

Cited By ~ 48

Author(s):

Changqing Zeng ◽

Susan M. Berget

Keyword(s):

Rna Polymerase Ii ◽

Repeat Sequence ◽

Splicing Factors ◽

Exon Definition ◽

Pol Ii ◽

Terminal Domain ◽

Nuclear Extracts ◽

In Vitro Splicing ◽

Precursor Rna

ABSTRACT Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3′ and 5′ splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.

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An Intronic Enhancer Regulates Splicing of the Twintron of Drosophila melanogaster prospero Pre-mRNA by Two Different Spliceosomes

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.1855-1869.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 1855-1869 ◽

Cited By ~ 28

Author(s):

Petra Scamborova ◽

Anthony Wong ◽

Joan A. Steitz

Keyword(s):

Drosophila Melanogaster ◽

Splicing Regulation ◽

Developmentally Regulated ◽

Opposite Pattern ◽

Enhancer Element ◽

Intronic Enhancer ◽

In Vitro Splicing ◽

Intron Region ◽

Sequence Requirements

ABSTRACT We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5′ splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.

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