The Mammalian pre-mRNA Splicing Apparatus

Lamond, Angus I.; Barabino, Silvia M.L.; Blencowe, B J

doi:10.1007/978-3-642-84150-7_15

Cited by 17 publications

(6 citation statements)

References 51 publications

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“…The major components of the spliceosome belong to the U-class of small nuclear ribonucleoprotein particles (U snRNPs), specifically Ul, U2, U4/U6 and U5 snRNPs. These four snRNPs, together with non-snRNP protein factors, assemble along an ordered pathway to form a functional spliceosome (for recent reviews see [1][2][3][4]. A combination of affinity selection methods and native gel analyses have shown that the U1, U2, U4/U6 and U5 snRNPs are present in splicing complexes (5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts

Lamm¹,

Blencowe²,

Sproat³

et al. 1991

Nucl Acids Res

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RNA duplexes containing the modified base 2-aminoadenine in place of adenine are stabilized through the formation of three hydrogen bonds in 2-amino A* U base pairs. Antisense 2'-0-alkyloligoribonucleotide probes incorporating 2-aminoadenosine are thus able to efficiently affinity select RNP particles which are otherwise inaccessible. This has allowed the efficient and specific depletion of U5 snRNP from HeLa cell nuclear splicing extracts. U5 snRNP is shown to be essential for spliceosome assembly and for both steps of pre-mRNA splicing. The absence of U5 snRNP prevents the stable association of U4/U6 but not Ul and U2 snRNPs with pre-mRNA. INTRODUCTIONThe splicing of nuclear messenger RNA precursors (pre-mRNA) occurs within a multicomponent structure termed the spliceosome. The major components of the spliceosome belong to the U-class of small nuclear ribonucleoprotein particles (U snRNPs), specifically Ul, U2, U4/U6 and U5 snRNPs. These four snRNPs, together with non-snRNP protein factors, assemble along an ordered pathway to form a functional spliceosome (for recent reviews see 1-4). A combination of affinity selection methods and native gel analyses have shown that the U1, U2, U4/U6 and U5 snRNPs are present in splicing complexes (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In order to study the roles of the different snRNP particles in the splicing mechanism, a variety of methods have been used to inactivate individual snRNP particles in in vitro splicing extracts. These include inhibition by snRNP-specific antibodies (15-17), site-specific cleavage of snRNA components by RNase H in the presence of complementary DNA oligonucleotides (17-21), and masking specific snRNA domains using antisense 2'-0-methyl oligoribonucleotides (2'-OMe RNA) (22 -24). In Saccharomyces cerevisiae it has also been possible to exploit a genetic approach in which expression of a specific snRNA gene is placed under the control of the inducible p3-gal promoter. This has allowed in vivo depletion of specific snRNP species (25 -27). Using one or more of the above methods, U 1,

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Section: Introductionmentioning

confidence: 99%

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts

Lamm¹,

Blencowe²,

Sproat³

et al. 1991

Nucl Acids Res

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“…T HE development of in vitro splicing extracts from mammalian and yeast systems has resulted in major advances in understanding the basic mechanism of pre-mRNA splicing (for reviews see references 9,27,38,41,54) . The splicing reaction involves two sequential transesterification reactions, similar to that seen with group II self-splicing introns (reviewed in reference 31) .…”

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confidence: 99%

Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies

Carmo‐Fonseca

Pepperkok

Carvalho

et al. 1992

The Journal of Cell Biology

388

262

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Abstract. We have recently shown that discrete foci are present in the nuclei of mammalian cells in which each of the Ul, U2, U4/U6, and U5 snRNPs involved in pre-mRNA splicing, and the non-snRNP-splicing factor U2AF, are concentrated (Carmo-Fonseca, M., D. Tollervey, R. Pepperkok, S. Barabino, A . Merdes, Here, we identify these snRNP-rich organelles as coiled bodies. snRNPs no longer concentrate in coiled bodies after cells are treated with the transcription inhibitors a-amanitin or actinomycin D. snRNP association with coiled bodies is also disrupted by heat shock . This indicates that the association of T HE development of in vitro splicing extracts from mammalian and yeast systems has resulted in major advances in understanding the basic mechanism of pre-mRNA splicing (for reviews see references 9, 27, 38, 41, 54) . The splicing reaction involves two sequential transesterification reactions, similar to that seen with group II self-splicing introns (reviewed in reference 31) . Unlike selfsplicing introns however, nuclear pre-mRNA splicing requires a complex set of trans-acting splicing factors which bind to substrate RNAs in an ordered pathway to form an active splicing complex or "spliceosome" (8, 17, 22, 26, 32, and 47) . Major components ofspliceosomes are the Ul, U2, U4/ U6, and U5 snRNPs, all ofwhich are required for both spliceosome assembly and splicing. In addition to these snRNNs, recent studies have identified a number of non-snRNP protein factors which are also required for splicing in vitro (4,23,25,33,34,55,69,74,75) .In contrast with the substantial progress in dissecting the splicing machinery in vitro, comparatively little is known about how the splicing factors are organized in vivo, or how splicing is integrated in the nucleus with transcription or RNA processing reactions such as 3' polyadenylation . Many previous studies have shown by indirect immunofluorescence that autoimmune and monoclonal antibodies specific for

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“…While the snRNAs U1, U2, and U5 are organized as separate ribonucleoprotein particles, the U4 and U6 snRNAs reside in the same RNP complex, forming the U4/U6 snRNP particle (Reddy and Busch, 1988). All four major snRNPs are essential trans-acting factors in the splicing of pre-mRNA (Steitz et al, 1988;Lamond et al, 1990;Liihrmann et al, 1990). Owing to the introduction of improved preparation techniques and the application of more sensitive analytical procedures, the number of well-characterized snRNP proteins has increased considerably during the last few years (Liihrmann et al, 1990).…”

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A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

Hackl

Fischer

Lührmann

1994

The Journal of Cell Biology

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Abstract. The biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, and U5 involves: (a) migration of the snRNA molecules from the nucleus to the cytoplasm; (b) assembly of a group of common proteins (Sm proteins) and their binding to a region on the snRNAs called the Smbinding site; and (c) translocation of the RNP back to the nucleus. A first prerequisite for understanding the assembly pathway and nuclear transport of the snRNPs in more detail is the knowledge of all the snRNP proteins that play essential roles in these processes.We have recently observed a previously undetected 69-kD protein in 12S U1 snRNPs isolated from HeLa nuclear extracts under non-denaturing conditions that is clearly distinct from the U1-70K protein.The following evidence indicates that the 69-kD protein is a common, rather than a Ul-specific, protein, possibly associating with the snRNP core particles by protein-protein interaction. (a) Antibodies raised against the 69-kD protein, which did not cross-react with any of the Sm proteins B'-G, precipitated not only U1 snRNPs, but also the other spliceosomal snRNPs U2, U4/U6 and U5, albeit to a lower extent. When U1 snRNA as well as a mutant U1 snRNA, that can bind the Sm core proteins but lacks the capacity to bind the Ul-specific proteins 70K, A, and C, were injected into Xenopus oocytes to allow assembly in vivo, they were recognized by antibodies specific against the 69-kD protein in the ooplasm and in the nucleus.The 69-kD protein is under-represented, if present at all, in purified 17S U2 and in 25S [U4/U6.U5] trisnRNPs, isolated from HeLa nuclear extracts. Our results are consistent with the working hypothesis that this protein may either play a role in the cytoplasmic assembly of the core domain of the snRNPs and/or in the nuclear transport of the snRNPs. After transport of the snRNPs into the nucleus, it may dissociate from the particles as for example in the case of the 17S U2 or the 25S [U4/U6.U5] tri-snRNP, which bind more than 10 different snRNP specific proteins each in the nucleus.

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The Mammalian pre-mRNA Splicing Apparatus

Cited by 17 publications

References 51 publications

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts

Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts

Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 snRNPs in coiled bodies

A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

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