Determinants of Nam8-dependent splicing of meiotic pre-mRNAs

Sloan-Kettering Institute, Weill Cornell Medical College, New York, NY 10065, USA.
Nucleic Acids Research (Impact Factor: 9.11). 04/2011; 39(8):3427-45. DOI: 10.1093/nar/gkq1328
Source: PubMed


Nam8, a component of yeast U1 snRNP, is optional for mitotic growth but required during meiosis, because Nam8 collaborates
with Mer1 to promote splicing of essential meiotic mRNAs AMA1, MER2 and MER3. Here, we identify SPO22 and PCH2 as novel targets of Nam8-dependent meiotic splicing. Whereas SPO22 splicing is co-dependent on Mer1, PCH2 is not. The SPO22 intron has a non-consensus 5′ splice site (5′SS) that dictates its Nam8/Mer1-dependence. SPO22 splicing relies on Mer1 recognition, via its KH domain, of an intronic enhancer 5′-AYACCCUY. Mutagenesis of KH and the enhancer
highlights Arg214 and Gln243 and the CCC triplet as essential for Mer1 activity. The Nam8-dependent PCH2 pre-mRNA has a consensus 5′SS and lacks a Mer1 enhancer. For PCH2, a long 5′ exon and a non-consensus intron branchpoint dictate Nam8-dependence. Our results implicate Nam8 in two distinct
meiotic splicing regulons. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments.
The leader, tail and RRM1 are dispensable for splicing meiotic targets and unnecessary for vegetative Nam8 function in multiple
synthetic lethal genetic backgrounds. Nam8 activity is enfeebled by alanine mutations in the putative RNA binding sites of
the RRM2 and RRM3 domains.

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    • "By conducting an analysis of the effects of C-terminal truncations of yeast Yhc1, we traced a wide network of genetic interactions between inessential segments of the Yhc1 C-terminal domain and all other components of the early spliceosome that were interrogated here, with the notable exception of Nam8. The nam8Δ allele is itself synthetic lethal or severely sick in the absence of Mud1, Mud2 or TMG caps, or when combined with benign mutations in U1 snRNA or an essential splicing factor (17,21,25,26,32–34,49), including, as shown here, with the N-terminal R21A mutation in Yhc1. The fact that nam8Δ has no effect when combined with the YHC1-(1-170), and that nam8Δ and YHC1-(1-170) have a similar spectrum of synergies with other spliceosome components, suggest that they are epistatic, i.e. "
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    ABSTRACT: Yhc1 and U1C are homologous essential subunits of the yeast and human U1 snRNP, respectively, that are implicated in the establishment and stability of the complex of U1 bound to the pre-mRNA 5' splice site (5'SS). Here, we conducted a mutational analysis of Yhc1, guided by the U1C NMR structure and low-resolution crystal structure of human U1 snRNP. The N-terminal 170-amino acid segment of the 231-amino acid Yhc1 polypeptide sufficed for vegetative growth. Although changing the zinc-binding residue Cys6 to alanine was lethal, alanines at zinc-binding residues Cys9, His24 and His30 were not. Benign alanine substitutions at conserved surface residues elicited mutational synergies with other splicing components. YHC1-R21A was synthetically lethal in the absence of Mud2 and synthetically sick in the absence of Nam8, Mud1 and Tgs1 or in the presence of variant U1 snRNAs. YHC1 alleles K28A, Y12A, T14A, K22A and H15A displayed a progressively narrower range of synergies. R21A and K28A bypassed the essentiality of DEAD-box protein Prp28, suggesting that they affected U1•5'SS complex stability. Yhc1 Arg21 fortifies the U1•5'SS complex via contacts with SmD3 residues Glu37/Asp38, mutations of which synergized with mud2Δ and bypassed prp28Δ. YHC1-(1-170) was synthetically lethal with mutations of all components interrogated, with the exception of Nam8.
    Full-text · Article · Feb 2014 · Nucleic Acids Research
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    • "The association of MER2 transcript weak 5′ splice site with U1, for example, is dependent on the interaction between Mer1p and U1 snRNP proteins during meiosis [45]. Mer1p binds to an intronic enhancer and mediates U1 snRNP association leading to spliceosome assembly on this substrate [45], [46], [47]. "
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    ABSTRACT: Splicing of primary transcripts is an essential process for the control of gene expression. Specific conserved sequences in premature transcripts are important to recruit the spliceosome machinery. The Saccharomyces cerevisiae catalytic spliceosome is composed of about 60 proteins and 5 snRNAs (U1, U2, U4/U6 and U5). Among these proteins, there are core components and regulatory factors, which might stabilize or facilitate splicing of specific substrates. Assembly of a catalytic complex depends on the dynamics of interactions between these proteins and RNAs. Cwc24p is an essential S. cerevisiae protein, originally identified as a component of the NTC complex, and later shown to affect splicing in vivo. In this work, we show that Cwc24p also affects splicing in vitro. We show that Cwc24p is important for the U2 snRNP binding to primary transcripts, co-migrates with spliceosomes, and that it interacts with Brr2p. Additionally, we show that Cwc24p is important for the stable binding of Prp19p to the spliceosome. We propose a model in which Cwc24p is required for stabilizing the U2 association with primary transcripts, and therefore, especially important for splicing of RNAs containing non-consensus branchpoint sequences.
    Full-text · Article · Sep 2012 · PLoS ONE
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    • "(Nam8 synthetic interactions are shown as gold lines in Figure 4.) Otherwise benign mutations of the Mud2-binding and Prp40-binding sites of Msl5 are lethal in a tgs1Δ background in which U1 snRNA lacks the TMG cap structure. (Tgs1 synthetic interactions are denoted by green lines in Figure 4.) These results, together with prior findings of synthetic lethality between tgs1Δ, nam8Δ, mud2Δ and mud1Δ (9,10,16,24), underscore the plentiful overlapping genetic connections between the Msl5-Mud2 heterodimer and the U1 snRNP. "
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    ABSTRACT: Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.
    Full-text · Article · Jan 2012 · Nucleic Acids Research
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