A Minimal Spliceosomal Complex A Recognizes the Branch Site and Polypyrimidine Tract

Query, Charles C.; McCaw, Patrick Schonleber; Sharp, Phillip A.

doi:10.1128/mcb.17.5.2944

Cited by 38 publications

(46 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The first ATPdependent step involves the formation of an A complex, respectively A3Ј complex (Konarska and Sharp, 1986;Query et al, 1997). In this respect, the results with mutant premRNAs correlate well with those performed at 4°C.…”

Section: Molecular Biology Of the Cell 402supporting

confidence: 73%

“…These RNAs form merely a minimal splicing complex Amin (Query et al, 1997). The RNAs also displayed some tendency to be targeted toward the speckles and their accumulations were often found adjacent to the speckles in the form of distinct dots or streaks.…”

Section: Molecular Biology Of the Cell 398mentioning

confidence: 99%

“…The AdBS-PPT RNA ( Figure 3B) and PIPBS-PPT RNA (our unpublished results), which both consist of just BS and PPT, generated a fluorescence pattern with RNAs staying outside of the speckles. These RNAs form merely a minimal splicing complex Amin (Query et al, 1997). The RNAs also displayed some tendency to be targeted toward the speckles and their accumulations were often found adjacent to the speckles in the form of distinct dots or streaks.…”

Section: Pre-mrna Structural Requirements For Its Targeting To the Spmentioning

confidence: 99%

“…Particularly important for the targeting to the speckles are the flanking downstream sequences to the 3Ј splice site. We emphasize that the targeting also takes place in the case of nonspliceable RNAs Ad10 and Ad11, which are able to form prespliceosome complexes in vitro (Frendewey and Keller, 1985;Christofori et al, 1987;Frendewey et al, 1987).It has been established that the E (early) complex formation (Seraphin and Rosbash, 1989;Bennett et al, 1992;Michaud and Reed, 1993) as well as complexes formed on the minimal RNA substrate consisting of BS and PPT sequence (called Amin complex) (Query et al, 1997) do not in vitro require ATP (Legrain et al, 1988;Michaud and Reed, 1991;Jamison et al, 1992;Query et al, 1997). The first ATPdependent step involves the formation of an A complex, respectively A3Ј complex (Konarska and Sharp, 1986;Query et al, 1997).…”

mentioning

confidence: 99%

“…RNAs containing a branch sequence and a PPT (BS-PPT) were transcribed from DNA templates generated by polymerase chain reaction. DNA template for PIPBS-PPT RNA (34 nucleotides, 5Ј-GGGUGCUGACUGGCU-UCUUCUCUCUUUUUCCCUC-3Ј [Query et al, 1997]) derived from pPIP85.B was obtained by polymerase chain reaction from the synthetic oligo (5Ј-TGCTGACTGGCTTCTTCTCTCTTTTTCCCTC-3Ј) by using oligonucleotides 77P9 (5Ј-TAATACGACTCACTAT-AGGGTGCTGACTGGCTTCTTC-3Ј), 77R0 (5Ј-GAGGGAAAAA-GAGAGAAG-3Ј), and DNA for AdBS-PPT RNA (44 nucleotides, 5Ј-GGGCCUUGAUGAUGUCAUACUUAUCCUGUCCCUUUUU-UUUCCAC-3Ј [Wansink et al, 1994]) derived from pSP62⌬i1 by using oligonucleotides 77R1 (5Ј-TAATACGACTCACTATAGGGCCTTGAT-GATGTCATAC-3Ј), 77R2 (5Ј-GTGGAAAAAAAAGGGAC-3Ј).All pre-mRNAs were transcribed either with SP6 or T7 RNA polymerase. The reaction mixture (25 l) contained a 1ϫ transcription buffer (Promega), 10 mM dithiothreitol, 1 g of linearized DNA template, 24 U RNasin, 1 mM GpppG, 50 M TMR-UTP, 20 U of either SP6 or T7 RNA polymerase in the presence of the nucleotide mixture consisting of 1 mM each of ATP and CTP, 100 M GTP, and 100 M UTP.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Melčák

Kopský

et al. 2001

MBoC

View full text Add to dashboard Cite

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed. INTRODUCTIONNuclear speckles are enriched in splicing factors and in the factors of the transcription machinery (Spector, 1990;Krause et al., 1994;Bregman et al., 1995;Larsson et al., 1995). Even though there is a consensus that these compartments play a role in RNA metabolism, their exact function is presently unknown. When growing mammalian cells are labeled with antibodies to splicing components, 20 to 50 shining nuclear domains, i.e., nuclear speckles, also termed SC35 domains (protein SC35 being an important serine/arginine (SR)-rich splicing factor [Fu and Maniatis, 1990]), splicing factor compartments, or just speckles, are usually observed (Perraud et al., 1979;Spector et al., 1983;Spector, 1990;Misteli, 2000). In some cell types, they occupy as much as 20% of the nuclear volume. At the electron microscope level, they consist of morphologically well-defined interchromatin granule clusters and of domains of perichromatin fibrils, some of which are believed to represent precursor-mRNAs (premRNAs) (Fakan and Puvion, 1980;Spector et al., 1983;Puvion et al., 1984;Spector, 1990;Fakan, 1994;Raška, 1995;Melčák et al., 2000).Most mammalian pre-mRNAs contain introns and typically have to be spliced before being transported to the cytoplasm. It has been shown biochemically that spliceosome formation and splicing may be cotranscriptional events (Wuarin and Schibler, 1994). More recent results indicate that transcription and splicing are coupled with interactions of certain factors in both processes. They take part in the large macromolecular c...

show abstract

Section: Molecular Biology Of the Cell 402supporting

confidence: 73%

Section: Molecular Biology Of the Cell 398mentioning

confidence: 99%

Section: Pre-mrna Structural Requirements For Its Targeting To the Spmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Melčák

Kopský

et al. 2001

MBoC

View full text Add to dashboard Cite

show abstract

Nuclear protein profiling of Jurkat cells during heat stress‐induced apoptosis by 2‐DE and MS/MS

et al. 2007

View full text Add to dashboard Cite

Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.

show abstract

Intronic features that determine the selection of the 3′ splice site

Pérez-Valle

Vilardell

2012

WIREs RNA

View full text Add to dashboard Cite

Most eukaryotic primary transcripts include segments, or introns, that will be accurately removed during RNA biogenesis. This process, known as pre‐messenger RNA splicing, is catalyzed by the spliceosome, accurately selecting a set of intronic marks from others apparently equivalent. This identification is critical, as incorrectly spliced RNAs can be toxic for the organism. One of these marks, the dinucleotide AG, signals the intronic 3′ end, or 3′ splice site (ss). In this review we will focus on those intronic features that have an impact on 3′ ss selection. These include the location and type of neighboring sequences, and their distance to the 3′ end. We will see that their interplay is needed to select the right intronic end, and that this can be modulated by additional intronic elements that contribute to alternative splicing, whereby diverse RNAs can be generated from identical precursors. This complexity, still poorly understood, is fundamental for the accuracy of gene expression. In addition, a clear knowledge of 3′ ss selection is needed to fully decipher the coding potential of genomes. WIREs RNA 2012 doi: 10.1002/wrna.1131 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > Splicing Regulation/Alternative Splicing Regulatory RNAs/RNAi/Riboswitches > Riboswitches

show abstract

A Minimal Spliceosomal Complex A Recognizes the Branch Site and Polypyrimidine Tract

Cited by 38 publications

References 68 publications

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Nuclear protein profiling of Jurkat cells during heat stress‐induced apoptosis by 2‐DE and MS/MS

Intronic features that determine the selection of the 3′ splice site

Contact Info

Product

Resources

About