scholarly journals The U1 snRNP Base Pairs with the 5′ Splice Site within a Penta-snRNP Complex

2003 ◽  
Vol 23 (10) ◽  
pp. 3442-3455 ◽  
Author(s):  
Hadar Malca ◽  
Noam Shomron ◽  
Gil Ast
Keyword(s):  
Splice Site ◽  
Regulatory Elements ◽  
Base Pairs ◽  
In Vitro System ◽  
Binding Reaction ◽  
Nuclear Extracts ◽  
U1 Snrnp ◽  
Splice Site Sequence ◽  
Precursor Mrna

ABSTRACT Recognition of the 5′ splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5′ splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5′ splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5′ splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5′ splice site through base pairing with the 5′ end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5′ splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5′ splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5′ splice site in a mammalian preassembled penta-snRNP complex.

Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

scholarly journals U1 small nuclear ribonucleoprotein studied by in vitro assembly.

1983 ◽  
Vol 96 (6) ◽  
pp. 1751-1755 ◽  
Author(s):  
E D Wieben ◽  
S J Madore ◽  
T Pederson
Keyword(s):  
Direct Analysis ◽  
Small Nuclear Rna ◽  
In Vitro System ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Specific Complex ◽  
Reticulocyte Lysate

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Effect of double-strand breaks on homologous recombination in mammalian cells and extracts.

1985 ◽  
Vol 5 (12) ◽  
pp. 3331-3336 ◽  
Author(s):  
K Y Song ◽  
L Chekuri ◽  
S Rauth ◽  
S Ehrlich ◽  
R Kucherlapati
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Recombination Frequency ◽  
Double Strand Breaks ◽  
Base Pairs ◽  
In Vitro System ◽  
Strand Breaks ◽  
Cell Extracts ◽  
Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

scholarly journals Expression of the murine alpha B-crystallin gene is not restricted to the lens.

1989 ◽  
Vol 9 (3) ◽  
pp. 1083-1091 ◽  
Author(s):  
R A Dubin ◽  
E F Wawrousek ◽  
J Piatigorsky
Keyword(s):  
Skeletal Muscle ◽  
Transcription Initiation ◽  
Germ Line ◽  
Initiation Site ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
In Vitro System ◽  
Endogenous Gene ◽  
Efficient Expression

The murine alpha B-crystallin gene was cloned and its expression was examined. In the mouse, significant levels of alpha B-crystallin RNA were detected not only in lens but also in heart, skeletal muscle, kidney, and lung; low and trace levels were detected in brain and spleen, respectively. The RNA species in lung, brain, and spleen was 400 to 500 bases larger than that in the other tissues. Transcription in lens, heart, skeletal muscle, kidney, and brain initiated at the same position. A mouse alpha B-crystallin mini-gene was constructed and was introduced into the germ line of mice, and its expression was demonstrated to parallel that of the endogenous gene. Transgene RNA was always detected in lens, heart, and skeletal muscle, while expression in kidney and lung was variable; it remains uncertain whether there is transgene expression in brain and spleen. These results demonstrate that regulatory sequences controlling expression of the alpha B-crystallin gene lie between sequences 666 base pairs upstream of the transcription initiation site and 2.4 kilobase pairs downstream of the poly(A) addition site and are not located within the introns. Transfection studies with a series of alpha B-crystallin mini-gene deletion mutants revealed that sequences between positions -222 and -167 were required for efficient expression in primary embryonic chick lens cells; sequences downstream of the poly(A) addition signal were dispensable for expression in this in vitro system.

scholarly journals Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

2010 ◽  
Vol 30 (8) ◽  
pp. 1878-1886 ◽  
Author(s):  
Martin J. Hicks ◽  
William F. Mueller ◽  
Peter J. Shepard ◽  
Klemens J. Hertel
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Base Pairing ◽  
Splice Site Selection ◽  
U1 Snrna ◽  
Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

scholarly journals p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

2002 ◽  
Vol 22 (15) ◽  
pp. 5443-5450 ◽  
Author(s):  
Zhi-Ren Liu
Keyword(s):  
Splice Site ◽  
Early Stage ◽  
Rna Helicase ◽  
Mrna Splicing ◽  
Cross Linking ◽  
Spliceosome Assembly ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
P68 Rna Helicase

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

scholarly journals Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4

1988 ◽  
Vol 8 (3) ◽  
pp. 1290-1300
Author(s):  
H Watanabe ◽  
T Imai ◽  
P A Sharp ◽  
H Handa
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Initiation Site ◽  
Base Pairs ◽  
Early Region ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Early Region 4

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

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