Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

scholarly journals Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

1988 ◽  
Vol 8 (4) ◽  
pp. 1558-1569 ◽  
Author(s):  
P E Cizdziel ◽  
M de Mars ◽  
E C Murphy
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Mrna Splicing ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Exon Ligation ◽  
Branch Point Sequence ◽  
Infected Cells ◽  
Low Efficiency

The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site, was studied. Cleavage at the 5' splice site in the revertant viral RNA proceeded in a temperature-sensitive fashion. No novel cryptic 3' splice sites were activated; however, splicing at an alternate upstream 3' splice site used at low efficiency in normal MuSVts110 RNA was increased to a level close to that of 5'-splice-site cleavage in the revertant viral RNA. Increased splicing at this site in 54-5A4 viral RNA is probably driven by the unavailability of the usual 3' splice site for exon ligation. The thermosensitivity of this alternate splice event suggests that the sequences governing the thermodependence of MuSVts110 RNA splicing do not involve any particular 3' splice site or branch point sequence, but rather lie near the 5' end of the intron.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

2018 ◽  
Author(s):  
Xiaofeng Zhang ◽  
Xiechao Zhan ◽  
Chuangye Yan ◽  
Wenyu Zhang ◽  
Dongliang Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Splice Site ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Exon Ligation ◽  
Cryo Electron Microscopy ◽  
Branch Point Sequence ◽  
Nuclear Rna ◽  
Before And After ◽  
Functional States

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

scholarly journals Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins

1998 ◽  
Vol 72 (7) ◽  
pp. 5493-5501 ◽  
Author(s):  
Siddhartha K. Biswas ◽  
Paul L. Boutz ◽  
Debi P. Nayak
Keyword(s):  
Influenza Virus ◽  
Protein Complex ◽  
Rna Synthesis ◽  
Sodium Dodecyl ◽  
Direct Interaction ◽  
Critical Factor ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Replication Assay

ABSTRACT Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the COOH terminus of NP. Analysis of NP deletion mutants revealed that at least three regions of NP interacted independently with PB2. A detailed analysis of the COOH terminus of NP by mutation of serine-to-alanine (SA) residues either individually or together demonstrated that SA mutations in this region did not affect the binding of NP to PB2. However, some SA mutations at the COOH terminus drastically affected the functional activity of NP in an in vivo transcription-replication assay, whereas others exhibited a temperature-sensitive phenotype and still others had no effect on the transcription and replication of the viral RNA. These results suggest that a direct interaction of NP with polymerase proteins may be involved in regulating the switch of viral RNA synthesis from transcription to replication.

scholarly journals RNA Splicing at Human Immunodeficiency Virus Type 1 3′ Splice Site A2 Is Regulated by Binding of hnRNP A/B Proteins to an Exonic Splicing Silencer Element

2001 ◽  
Vol 75 (18) ◽  
pp. 8487-8497 ◽  
Author(s):  
Patricia S. Bilodeau ◽  
Jeffrey K. Domsic ◽  
Akila Mayeda ◽  
Adrian R. Krainer ◽  
C. Martin Stoltzfus
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Splice Site ◽  
Rna Splicing ◽  
Consensus Sequence ◽  
Mrna Levels ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of exonic splicing silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of splicing at 3′ splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro splicing assays, we show that ESSV represses splicing when placed downstream of a heterologous splice site. A1, A1B, A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore splicing repression. The results suggest that coordinate repression of HIV-1 RNA splicing is mediated by members of the hnRNP A/B protein family.

scholarly journals A Flexible RNA Backbone within the Polypyrimidine Tract Is Required for U2AF65 Binding and Pre-mRNA Splicing InVivo

2010 ◽  
Vol 30 (17) ◽  
pp. 4108-4119 ◽  
Author(s):  
Chun Chen ◽  
Xinliang Zhao ◽  
Ryszard Kierzek ◽  
Yi-Tao Yu
Keyword(s):  
Molecular Biology ◽  
Splice Site ◽  
Negative Impact ◽  
Mrna Splicing ◽  
Polypyrimidine Tract ◽  
Backbone Flexibility ◽  
Naturally Occurring ◽  
Splicing Defect ◽  
Rna Backbone

ABSTRACT The polypyrimidine tract near the 3′ splice site is important for pre-mRNA splicing. Using pseudouridine incorporation and in vivo RNA-guided RNA pseudouridylation, we have identified two important uridines in the polypyrimidine tract of adenovirus pre-mRNA. Conversion of either uridine into pseudouridine leads to a splicing defect in Xenopus oocytes. Using a variety of molecular biology methodologies, we show that the splicing defect is due to the failure of U2AF65 to recognize the pseudouridylated polypyrimidine tract. This negative impact on splicing is pseudouridine specific, as no effect is observed when the uridine is changed to other naturally occurring nucleotides. Given that pseudouridine favors a C-3′-endo structure, our results suggest that it is backbone flexibility that is key to U2AF binding. Indeed, locking the key uridine in the C-3′-endo configuration while maintaining its uridine identity blocks U2AF65 binding and splicing. This pseudouridine effect can also be applied to other pre-mRNA polypyrimidine tracts. Thus, our work demonstrates that in vivo binding of U2AF65 to a polypyrimidine tract requires a flexible RNA backbone.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Interaction between the Negative Regulator of Splicing Element and a 3′ Splice Site: Requirement for U1 Small Nuclear Ribonucleoprotein and the 3′ Splice Site Branch Point/Pyrimidine Tract

1999 ◽  
Vol 73 (3) ◽  
pp. 2394-2400 ◽  
Author(s):  
Craig R. Cook ◽  
Mark T. McNally
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Rna Splicing ◽  
Nuclear Extract ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
A Minor

ABSTRACT The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several ciselements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5′ splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3′ splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3′ splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3′ splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3′ splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3′ splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2

1985 ◽  
Vol 5 (9) ◽  
pp. 2405-2413
Author(s):  
B M Bhat ◽  
H A Brady ◽  
W S Wold
Keyword(s):  
Steady State ◽  
Splice Site ◽  
Transcription Unit ◽  
Mrna Splicing ◽  
Deletion Mutants ◽  
Splice Sites ◽  
Rich Region ◽  
Specific Sequence ◽  
Exon Deletion

Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region.

scholarly journals Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

2003 ◽  
Vol 77 (3) ◽  
pp. 2105-2115 ◽  
Author(s):  
Xuefeng Liu ◽  
Akila Mayeda ◽  
Mingfang Tao ◽  
Zhi-Ming Zheng
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Viral Rna ◽  
Splicing Factor ◽  
Rna Expression ◽  
Bovine Papillomavirus ◽  
Phosphatidylinositol 3 Kinase ◽  
Selection Of ◽  
Phosphatidylinositol 3

ABSTRACT Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

Sign in / Sign up

Export Citation Format

Share Document