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...

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.

Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an identical mechanism to mRNA splicing, although there is no general requirement for either proteins or co-factors. Thus it seems likely that the Group 2 and nuclear mRNA splicing reactions have diverged from a common ancestor. tRNA genes are also interrupted by introns, but here the splicing mechanism is quite different because it is catalyzed by three enzymes, all proteins and with an intrinsic requirement for ATP hydrolysis.tRNA splicing occurs in all three major lines of descent, the Bacteria, the Archaea, and the Eukarya. In bacteria the introns are self-splicing (1-3). Until recently it was thought that the mechanisms of tRNA splicing in Eukarya and Archaea were unrelated as well. In the past year, however, it has been found that the first enzyme in the tRNA splicing pathway, the tRNA endonuclease, has been conserved in evolution since the divergence of the Eukarya and the Archaea. Surprising insights have been obtained by comparison of the structures and mechanisms of tRNA endonuclease from these two divergent lines. tRNA Precursors in Eukarya and ArchaeaThe earliest studies of tRNA splicing were in the yeast Saccharomyces cerevisiae where tRNA introns were first discovered (4, 5). With the completion of the S. cerevisiae genome sequence it is now known that yeast contains 272 tRNA genes of which 59, encoding 10 different tRNAs, are interrupted by introns (6). The introns are 14 -60 nucleotides in length and interrupt the anticodon loop immediately 3Ј to the anticodon (7). Among the 10 different yeast pre-tRNAs there is no conservation of sequence at the splice junctions although the 3Ј-splice junction is invariably in a bulged loop (8). Early studies on the structure of yeast tRNA precursors showed that the conformation of the mature domain is retained suggesting the model of the tertiary structure of eukaryal pre-tRNA shown in Fig. 1A (9, 10).In the Archaea the introns are also small and often interrupt the anticodon loop, but they are found elsewhere as well, for example interrupting the dihydro U stem (11). In several of the Archaea, tRNA genes have been found that contain two introns. The splice sites are found in an absolutely conserved structural motif consisting of two loops of three bases separated by a four-base pair helix, the bulge-helix-bulge (BHB) 1 motif (12). This structure, modeled in Fig. 1B from the related TAR RNA structure (13), allows the archaeal splicing mechanism to be extended to introns in rRNA that also retain this motif. Thus, early on it was suggested that the eukaryal and archaeal splicing sys...

In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing. A bank of ~1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe. We isolated 16 mutants defining 11 new complementation groups prp(ma)17-prp(ma}27 with four phenotypic classes of mutants and 21 mutants in the prp2-prpll complementation groups (formerly ma2-mall]. The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(ma}2-prp(ma)ll mutants. Three novel classes of mutants were isolated in this study. One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate oi splicing, with no change in the levels of pre-mRNA. The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing. The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron. All mutants isolated were recessive for the splicing phenotype. Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive. This study, together with previous isolation of the prp(ma)2-prp(ma)ll groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing. The precise removal of the intron from nuclear premRNA is an essential process in eukaryotic gene expres sion. The splicing reaction takes place in a complex par ticle termed the spliceosome (Brody and Abelson 1985;Frendewey and Keller 1985;Grabowski et al. 1985). The fimction of the spliceosome in nuclear pre-mRNA splicing is to align the splice sites and to catalyze the two-step splicing reaction. Therefore, to understand mRNA splicing, it will not only be necessary to eluci date the components and their functions but also the pathway of assembly of the spliceosome.The process of pre-mRNA splicing appears to be very similar in the yeast and the mammalian systems, but a particular advantage to the study of splicing in yeast is the facility with which a genetic approach can be applied to this problem. Not only can mutants defective in splicing be used to enumerate the components and their interactions but they can be useful in delineating steps in the assembly of the spliceosome. Before the process of pre-mRNA splicing was discovered, a set of tempera ture-sensitive mutants in RNA synthesis was found that defined the RNA2-RNA11 genes (Hartwell 1967). Sur prisingly, all of these mutants are defective in mRNA splicing (for review, see Warner 1987; Vija3nraghavan and Abelson 1989). By general consensus among the commu nity of researchers working on RNA processing in yeast and the yeast genetic stock center, these mutants will henceforth be called pre-RNA processing [prp] mutants.

The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla. In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA. We have cloned the genes encoding all four protein subunits of endonuclease. Each gene is essential. Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids. This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other Archaea. Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively. The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.

The Saccharomyces cerevisiae genes PRP2, PRP16, and PRP22 encode pre-mRNA splicing factors that belong to the highly conserved ''DEAH'' family of putative RNA helicases. We previously identified two additional members of this family, JA1 and JA2. To investigate its biological function, we cloned the JA1 gene and generated alleles carrying mutations identical to those found in highly conserved regions of other members of the DEAH family. A ja1 allele carrying a mutation identical to that in the temperaturesensitive (ts) prp22-1 gene conferred ts phenotype when integrated into the genome of a wild-type strain by gene replacement. Northern analysis of RNA obtained from the ts strain shifted to a nonpermissive temperature revealed accumulation of unspliced pre-mRNAs and excised intron lariats. Furthermore, analysis of splicing complexes showed that intron lariats accumulated in spliceosomes. The results presented indicate that JA1 encodes a pre-mRNA processing factor (Prp) involved in disassembly of spliceosomes after the release of mature mRNA. We have therefore renamed this gene PRP43.Numerous genes from Escherichia coli to humans encode proteins that belong to a highly conserved superfamily that comprises many well characterized DNA helicases and putative RNA helicases. Proteins encoded by these genes share extensive homology over a 300-amino acid domain containing seven highly conserved motifs (reviewed in refs. 1 and 2). These proteins can be grouped into families according to the sequences of the seven motifs. For example, the ''DEAD-box'' family is characterized by the amino acid sequence D-E-A-D in motif II, while the ''DEAH-box'' family, also named after its invariable sequence in motif II, has an additional 300-amino acid C-terminal domain present only in DEAH-box proteins.Although specific functions of the putative RNA helicases in pre-mRNA splicing have not yet been elucidated at the molecular level, it is likely that these proteins modulate the network of RNA-RNA interactions that occur during spliceosome assembly and splicing of pre-mRNA (3, 4). For example, in the early stages of spliceosome assembly the U4 and U6 small nuclear RNAs (snRNAs) are extensively base paired to form a double-stranded structure that is completely dissociated before the first catalytic step of splicing (5-7). Consistent with the need of energy to unwind the double-stranded structure, the DEIH-box protein Slt22͞Brr2 has been implicated in the dissociation of the U4͞U6 small nuclear ribonucleoprotein particle (snRNP) (P. Raghunathan and C. Guthrie, personal communication) as well as in the regulation of U2, U6, and U5 snRNA interactions (8). The DEAH-box factor Prp16 has been proposed to mediate conformational changes in the spliceosome (9) and to play a proofreading role in selecting the proper splice sites and branch point (10). The DEAD-box protein Prp5 has been proposed to mediate an ATP-dependent conformational change in the U2 snRNP prior to binding to the branch site of pre-mRNA (3,11,12). Furthermore, an ATP-dep...

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.