Spliceosome assembly in yeast.

Abstract

Downloaded from 5p11ce050me a55em61y 1n yea5t 5nRNA5 have 6een 5h0wn t0 6e a550c1ated w1th the 5p11-ce050me (P1k1e1ny et a1. 1986., 2. H05t0m5ky et a1., unpu61.; N. R1ede1 et a1., per5. c0mm.}, and we 5ha11 pr0-v1de further ev1dence f0r 5uch a550c1at10n5 1n th15 paper. 7he c011ect10n 0f yea5t 5nRNA5 115ted 1n 7a61e 1 have 6een further 1mp11cated 1n mRNA 5p11c1n9 6ecau5e they are prec1p1tated 6y a human aut01mmune ant15erum 5m 1M. J0ne5 and C. 6uthr1e, per5. c0mm.} a5 are the1r mamma11an c0unterpart5. 7h15 ant15erum rec09n12e5 a pr0te1n c0mp0nent wh1ch 15 c0mm0n t0 a11 f1ve U5nRNP5 1nv01ved 1n 5p11c1n9. 7he pr0te1n5 re4u1red f0r mRNA 5p11c1n9 have 6een 1e55 5tud1ed, 6ut 1n yea5t a 5et 0f we11-5tud1ed 9ene5, the RNA 9ene5, enC0de pr0te1n c0mp0nent5 0f the mRNA 5p11c1n9 apparatu5 (Lu5t19 et a1. 1986}. A yea5t t5 5tra1n mutant 1n 0ne 0f the5e 9ene5, ma2, ha5 pr0v1ded 50me 1nf0rmat10n a60ut the a55em61y 0f the 5p11ce050me.U51n9 the rna2 mutant extract, we have 5h0wn that the 5p11ce050me 15 a true 1ntermed1ate 1n the 5p11c1n9 pathway (L1n et a1. 1987}. 7he5e exper1ment5 a150 dem0n5trate that extr1n51c pr0te1n5 act 0n the fu11y a55em-61ed 5p11ce050me. We w111 further 5h0w 1n th15 paper that heat-1nact1vated rna2 mutant extract5 accumu1ate an 1dent1f1a61e a55em61y 1ntermed1ate. 5p11c1n9 c0mp1exe5 can 6e re501ved 6y e1ectr0ph0re515 1n n0ndenatur1n9 p01yacry1am1de 9e15 (K0nar5ka and 5harp 1986; P1k1e1ny et a1. 1986; K0nar5ka and 5harp 1987). 1n th15 paper we u5e that techn14ue t0 1dent1fy f0ur 5p11c1n9-5pec1f1c c0mp1exe5 1n the a55em61y 0f the yea5t 5p11ce050me. U51n9 the 5ame 9e1 5y5tem, we have d15c0vered that three 0f the 5nRNA5 f0und 1n the5e c0mp1exe5, 5nR6, 5nR7, and 5nR14, a550c1ate 1n extract5 t0 f0rm a 1ar9e c0mp1ex. 7h15 c0mp1ex under90e5 an A7P-dependent d1550c1at10n. Re5u1t51dent1f1cat10n 0f tw0 5p11c1n9 c0mp1exe5, A and 8 70 5tudy the f0rmat10n 0f the 5p11ce050me, we have fract10nated 5p11c1n9 react10n m1xture5 0n n0ndena-tur1n9 p01yacry1am1de 9e15. 7he 5u65trate5 1n the5e exper1ment5 were a2P-1a6e1ed 5P6 RNA p01ymera5e tran5cr1pt5 0f a p0rt10n 0f the yea5t act1n 9ene. 5u65trate5 c0nta1n1n9 mutat10n5 1n the act1n 1ntr0n were a150 u5ed. 7he5e mutat10n5 were c0n5tructed 1n a 6ack9r0und 1n wh1ch a crypt1c 7AC7AAC 5e4uence had 6een de1eted 1A6 act1n) (V1jayra9havan et a1. 19861. Hepar1n wa5 added t0 term1nate the 5p11c1n9 react10n and t0 e11m1nate n0n-5pec1f1c 61nd1n9 0f pr0te1n5 t0 the RNA5 (K0nar5ka and 5harp 1986}.1n1t1a11y, tw0 c0mp1exe5 were 065erved dur1n9 the c0ur5e 0f the react10n {F19. 1A). C0mp1ex 8 appeared after 1 m1n 0f 1ncu6at10n f0110wed 6y c0mp1ex A. 80th 0f the5e c0mp1exe5 are 5pec1f1c f0r 5p11c1n9 6ecau5e ne1ther f0rmed w1th an ant15en5e act1n tran5cr1pt 0r w1th RNA c0nta1n1n9 an a1tered 5• 5p11ce 51te (A1 0r C1} 0r 7AC-7AAC 5e4uence (A256, A257, 0r C259). W1th the 3• 5p11ce 51te mutat10n {ACAC}, the f1r5t 5tep 0f 5p11c1n9 can 0ccur 6ut the 5ec0nd 5tep 15 610cked. 7h15 5u65trate can f0rm 5p11ce050me5 a5 detected 6y 91ycer01 9rad1ent 5ed1mentat10n {V1jayra9havan et a1. 1986}. When th15 5u65tr...

Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition

“…The presence of crn-like TPR elements in several RNAprocessing proteins (McLean & Rymond, 1998) suggested that Clf1p might also contribute to pre-mRNA splicing+ To test this, the efficiency of yeast pre-mRNA splicing was monitored as a function of time after transcriptional repression of GAL1::CLF1+ Metabolic depletion of Clf1p clearly inhibited pre-mRNA splicing as RP51A (and ACT1, see below) mRNA levels dropped and pre-mRNA levels increased with incubation in the glucose-based medium (Fig+ 3A)+ The time course of splicing impairment and the subsequent growth arrest were indistinguishable from what has been previously reported for other GAL fusions, such as PRP8 (Brown & Beggs, 1992) and the genes for TPR proteins Prp39p (Lockhart & Rymond, 1994) and Prp42p (McLean & Rymond, 1998)+ No Clf1p-dependent changes in RNA mobility were observed with several intron-free pol II transcripts, including the U2 snRNA (Fig+ 3B)+ Primer extension analysis confirmed that the more slowly migrating RNAs observed with RP51A and actin (ACT1) hybridization probes were largely due to increased levels of pre-mRNA and not the similarly sized lariat intermediate (Fig+ 4 and data not shown)+ From these results we conclude that the growth arrest observed after GAL1::CLF1 repression results from a defect in cellular pre-mRNA splicing+ Extracts prepared from yeast cultures depleted of Clf1p were unable to process exogenously added premRNA (Fig+ 5A)+ This splicing deficiency was associated with a specific defect in spliceosome assembly (Fig+ 5B)+ When splicing reactions were resolved by native polyacrylamide gel electrophoresis, pre-mRNA from the Clf1p-complete extract was rapidly assembled into the U1, U2-containing prespliceosome band (complex A, Fig+ 5B)+ As expected based on previous studies (Pikielny et al+, 1986;Cheng & Abelson, 1987), the prespliceosome was converted with time into the more slowly migrating, snRNP-complete spliceosome band (complex B, Fig+ 5B)+ In contrast to the wildtype extract, a single splicing complex band formed in the Clf1p-depleted extract that comigrated with the well-characterized prespliceosome complex+ Prespliceosome arrest is clearly not a de facto consequence of inhibited growth or pre-mRNA splicing, as depletion of other essential splicing factors block assembly at earlier or later times in assembly (e+g+, see Lockhart & Rymond, 1994;McLean & Rymond, 1998;Xie et al+, 1998)+ The time of appearance and level of abundance of the putative Clf1-defective presplice- osome were similar to that observed for the wild-type prespliceosome, indicating that Clf1p-depletion did not significantly impair earlier steps in splicing complex assembly+…”

“…The native gel assay results suggested that tri-snRNP addition was impaired in the Clf1p-depleted extract+ However, because the electrophoretic mobility of a Clf1p-deficient spliceosome cannot be predicted with certainty, it remained possible that the U4, U5, or U6 snRNA bound in the absence of Clf1p+ If so, then the Clf1p-defective complex would need to be viewed as more elaborate than the normal prespliceosome+ To address this issue, splicing complexes were affinity purified on biotin-substituted pre-mRNA and then assayed for snRNA content by Northern blot (Fig+ 6)+ When a Clf1HAp-complete control extract was used, each of the spliceosomal snRNAs was recovered with the biotinylated RP51A pre-mRNA (Fig+ 6, lane 4)+ As previously observed (Pikielny et al+, 1986;Cheng & Abelson, 1987;Konarska & Sharp, 1987;Xie et al+, 1998), the U4 snRNA was underrepresented in the mature splicing complexes (Fig+ 6, lane 4) relative to the unfractionated extract (Fig+ 6, lane 1) due to U4 release prior to 59 splice site cleavage+ The U1 and U2 snRNAs, but few U4, U5, or U6 snRNAs, were recovered from splicing complexes assembled in the Clf1p-depleted extract (Fig+ 6, lane 3)+ Equivalent snRNA profiles were observed with earlier (5 and 15 min) time points of assembly in the Clf1p-depleted extract (data not shown)+ In all cases, however, the U1 and U2 snRNA recovery was substrate dependent, as virtually no snRNA copurified with a nonbiotinylated control substrate RNA (Fig+ 6, lane 5) or on RNAs lacking splice sites (Rymond et al+, 1987 and data not shown)+ The low level of tri-snRNP-derived snRNA in the Clf1p-defective complexes was not due to general degradation in the extract, as the U4, U5, and U6 snRNAs were abundantly present in the unbound fraction (Fig+ 6, lane 2)+ Together with the data presented above these results show Phenotypic assay of growth in the presence or absence of Clf1p+ Yeast cultures containing the wild-type CLF1 gene (CLF1) and the clf1::HIS3 disruptant transformed with GAL1::CLF1 or its viable mutant derivative GAL1::clf1(679) were streaked on galactose-based rich medium (GAL) or glucose-based medium (GLU) and incubated at 30 8C+ C: Complementation by the Drosophila crn gene+ Wild-type yeast (CLF1) and the clf1::HIS3 disruptant transformed with TEF::crn were streaked on YPD medium and incubated at 30 8C+ FIGURE 3. Analysis of cellular pre-mRNA splicing after Clf1p-depletion+ A: Wild-type yeast (CLF1) and the GAL1:CLF1 yeast culture were grown continuously on galactose (T ϭ 0) or shifted to glucose-based medium for the indicated times+ Total cellular RNA was extracted at each time point and then resolved on a denaturing agarose-formaldehyde gel+ A: The results of hybridization with the intron-containing RP51A probe+ The positions of the pre-mRNA and mRNA are schematically represented at the left of the image+ B: The same filter after hybridization with the SNR20 U2 snRNA gene+ that the U4/U6+U5 tri-snRNP particle does not bind productively to the prespliceosome in the absence of Clf1p+ Weak or transient interactions between the trisnRNP and prespliceosome might occur without Clf1p, but such interactions are insufficient to support premRNA splicing in vivo or in vitro+ Splicing in the Clf1p-depleted extracts was partially reconstituted with a hemagglutinin-tagged Clf1p protein (ClfHAp) synthesized in a rabbit reticulocyte lysate ( The results presented above suggest that Clf1p likely promotes spliceosome assembly through TPR-based interactions that help organize the U4/U6+U5 tri-snRNP particle or help tether this particle to the prespliceosome+ Consistent with the latter suggestion, twohybrid studies revealed that Clf1p interacts with at least two components of the yeast commitment complex a...…”

Pseudouridines in spliceosomal snRNAs

“…Splicing is catalyzed by the spliceosome, a large, dynamic RNA-protein complex that takes on various forms through the different steps of splicing (Konarska and Sharp, 1986;Cheng and Abelson, 1987;Nilsen, 1994;Burge et al, 1999;Yu et al, 1999;Karijolich and Yu, 2008;Will and Lührmann, 2011). Spliceosome activity depends on a large number of protein components, as well as five "uridyl-rich" snRNAs-U1, U2, U4, U5, and U6.…”

Schizosaccharomyces pombe U4 small nuclear RNA closely resembles vertebrate U4 and is required for growth

“…The high evolutionary conservation of U6 and the release (or less-tight association) of U4 from the spliceosome prior to intron cleavage (Pikielny et al, 1986;Cheng & Abelson, 1987;Lamond et al, 1988) has led to the further suggestion that U6 is the enzymically active component, and that the role of V4 is to act as a carrier for U6 and to block its activity until the appropriate moment. However, it is notable that only a part of the region of highest primary sequence conservation between V4 from Schizo pombe and vertebrates corresponds to the V4jU6 interaction domain.…”

“…Major events in spliceosome assembly include the association of UI and U2 small nuclear ribonucleoproteins (snRNPs) with the 5' splice junction and site of branch formation, respectively. U4 and U6 snRNA are associated by hydrogen-bonding and are found in a single snRNP particle (Bringmann et al, 1984;Hashimoto & Steitz, 1984) and may associate with the spliceosome as a U4/U5/U6 complex; U4 is released from the spliceosome before intron cleavage (Pikielny et al, 1986;Cheng & Abelson, 1987;Lamond et al, 1988). The site and mechanism of action of U4 snRNP remains obscure but it is essential for splicing activity in vitro.…”