RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells

Lelay-Taha, Marie-Noëlle; Reveillaud, Isabelle; Brunel, Joannes Sri-Widada Claude; Jeanteur, Philippe

doi:10.1016/0022-2836(86)90321-9

Cited by 63 publications

(21 citation statements)

References 44 publications

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“…The U1 snRNP-enriched fraction was prepared in two steps: First, a concentrated U snRNP fraction was obtained by isopycnic centrifugation of a HeLa nuclear extract in cesium chloride at 15 mM magnesium (Lelay-Taha et al 1986); second, pooled fractions containing U1 snRNP were loaded onto a DEAE column and eluted with a linear gradient of ammonium chloride. Fraction 18, which was used for the complementation assay, contained concentrated U1 snRNP and was devoid of the other U snRNPs, with the exception of a trace level of U6 (Fig.…”

Section: Targeted Binding Of P61 Is Governed By U1 Snrnpmentioning

confidence: 99%

U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

Hoffman¹,

Grabowski²

1992

Genes & Development

198

154

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A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5' splice site of E4 is coincident with the selection of the 3' splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3' splice site of E4 in a manner that is clearly facilitated by a downstream 5' splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3' splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.[Key Words: RNA-protein binding; U1 snRNP; U2AF65; alternative splicing; exon selection] Received May 8, 1992; revised version accepted October 2, 1992.Alternative splicing of pre-mRNA is a widespread process that is largely responsible for the diversity of polypeptides expressed in mammalian cells, yet a molecular understanding of the mechanisms and factors that govern this process is lacking. A central question of interest is how to explain the regulation of splice site selection that leads to the observed patterns of alternative 5', or 3' splice site selection, mutually exclusive exon selection, or controlled exon skipping (Andreadis et al. 1987;Maniatis 1991).Our current understanding, from studies of single iniron-containing pre-mRNA substrates, is that splice site recognition is achieved by the assembly of a spliceosome complex. The spliceosome is known to contain the small nuclear ribonucleoprotein (snRNP) particles U1, U2, U5, and U4 + U6, as well as additional protein factors Steitz et al. 1988;Green 1991;Guthrie 1991). In one of the earliest spliceosome assembly events, the 5' splice site at the upstream boundary of the intron is recognized by direct base-pairing with a 6-to 9-nucleotide stretch at the 5' end of U1 small nuclear ~Affifiated with btCB Graduate Program, Brown University, Providence, Rhode Island 02912 USA. 2Corresponding author.RNA (snRNA) (Zhuang and Weiner 1986;Siliciano and Guthrie 1988;Rosbash and Seraphin 1991). This RNA base-pairing interaction appears to be further stabilized by the involvement of a U1 snRNP-specific protein, U1-C (Heinrichs et al. 1990). Interestingly, engineered nucleotide changes in the 5' end of U1 snRNA have been shown to alter 5' splice s...

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Section: Targeted Binding Of P61 Is Governed By U1 Snrnpmentioning

confidence: 99%

U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

Hoffman¹,

Grabowski²

1992

Genes & Development

198

154

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“…The presence of Ull snRNA was thus not strictly correlated with the processing activities. SF-containing fractions (30-34) were pooled and subjected to CsCl buoyant density centrifugation in the presence of 15 niM MgCl2 and 5 mM DTT to stabilize snRNPs during centrifugation (Reveillaud et al 1984;Lelay-Taha et al 1986). As shown in Figure 7, B and C, both cleavage and polyadenylation activities were again detected in exactly the same fractions (11-13), at a density of 1.27-1.30 g/ml.…”

Section: Characterization Of the Specificity Factormentioning

confidence: 99%

“…To 2 ml of SF-containing fractions obtained from two phenyl Superose chromatographies (2.5 mg of protein), MgClj, and DTT were added to final concentrations of 15 and 5 mM, respectively, to stabilize snRNPs during centrifugation (Reveillaud et al 1984;Lelay-Taha et al 1986). Solid CsCl was then added to a final concentration of 40% (wt/wt).…”

Section: Fractionation Of Factors Involved In Pre-mrna 3'-end Cleavagmentioning

confidence: 99%

Four factors are required for 3'-end cleavage of pre-mRNAs.

Takagaki¹,

Ryner²,

Manley³

1989

Genes Dev.

202

174

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We reported previously that authentic polyadenylation of pre-mRNAs in vitro requires at least two factors: a cleavage/specificity factor (CSF) and a fraction containing nonspecific poly(A) polymerase activity. To study the molecular mechanisms underlying 3' cleavage of pre-mRNAs, we fractionated CSF further and show that it consists of four separable subunits. One of these, called specificity factor (SF; M" -290,000), is required for both specific cleavage and for specific polyadenylation and thus appears responsible for the specificity of the reaction. Although SF has not been purified to homogeneity, several lines of evidence suggest that it may not contain an essential RNA component. Two other factors, designated cleavage factors I (CFI; M" -130,000) and II (CFII; M" -110,000), are sufficient to reconstitute accurate cleavage when mixed with SF. A fourth factor, termed cleavage stimulation factor (CstF; M" -200,000), enhances cleavage efficiency significantly when added to a mixture of the three other factors. CFI, CFII, and CstF do not contain RNA components, nor do they affect specific polyadenylation in the absence of cleavage. Although these four factors are necessary and sufficient to reconstitute efficient cleavage of one pre-RNA tested, poly(A) polymerase is also required to cleave several others. A model suggesting how these factors interact with the pre-mRNA and with each other is discussed.[Key Words: Cleavage/specificity factor; poly(A); pre-RNA]

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“…Binding of an antisense 2'-O-methyl (2'-OMe) RNA oligonucleotide in the same region, however, did not result in a defect of in vitro splicing (8). Binding of the common snRNP proteins (Sm proteins) involves the 3'-terminal stem-loop, the Sm-binding site (nucleotides 120 to 126), and the central stem-loop (27). In analogy to the U2 snRNP (30, 31), Sm protein binding of the U4 RNA is believed to be necessary for cytoplasmic cap trimethylation and nuclear snRNP transport; the m3G cap and the Sm domain together are considered a bipartite nuclear localiza-…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

Wersig¹,

Bindereif²

1992

Mol. Cell. Biol.

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We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro.Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.Nuclear pre-mRNA splicing requires the ordered assembly of the splicing substrate into a large ribonucleoprotein (RNP) complex, the spliceosome, containing small nuclear RNPs (Ul, U2, U4/U6, and U5 snRNPs) and non-snRNP splicing factors (for recent reviews, see references 6, 28, and 40). The Ul and U2 snRNPs, in conjunction with nonsnRNP splicing factors, recognize the 5' splice site and the branch point region, respectively. Most likely, the U4/U6 and U5 snRNPs interact to form the U4/U5/U6 multi-snRNP before they are incorporated into the spliceosome (4, 25, 37), apparently not through direct pre-mRNA contacts but through snRNP-snRNP interactions (5). The spliceosome proceeds, during its assembly, the two steps of the splicing reaction, and its disassembly, through multiple conformational stages, some of which may be driven by ATP-dependent RNA helicases (for reviews, see references 17 and 36). For example, a major conformational change during splicing destabilizes U4 binding in the spliceosome (8,11,26,35); U6 and U2 RNAs engage in a base-pairing interaction required for the assembly of a stable spliceosome and for splicing (12,21,46,47), and an additional U6-U2 interaction has recently been suggested (32).On the basis of extensive phylogenetic evidence, a secondary structure model has been proposed for U4 RNA base paired with U6 RNA (18) (for the human U4/U6 RNA hybrid, see Fig. 1). U4 RNA can be divided into three domains, the 5'-terminal U4-U6 interaction domain (stem II, 5' stem-loop, stem I), the central domain (single-stranded region, central stem-loop), and the 3'-terminal domain (Smbinding site, 3' stem-loop). Interestingly, free U4 RNA can also be folded into a conserved secondary structure, in which the 3'-terminal domain remains unchanged compared with the U4/U6 RNA hybrid structure but in which the stem I and stem II regions are partially base paired with each other, thereby extending the 5' stem-loop structure (33). There are several lines of indirect evidence that U6 RNA sequences play a catalytic role in splicing (10, 14, 42, 43; reviewed in reference 17). Since in the U4/U6 snRNP U6 * Corresponding author.RNA is stably associated with U4 RNA, U4 has been hypothesized to function as an antisense negative ...

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RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells

Cited by 63 publications

References 44 publications

U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon.

Four factors are required for 3'-end cleavage of pre-mRNAs.

Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

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