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Structure-function analysis of the 5' end of yeast U1 snRNA highlights genetic interactions with the Msl5*Mud2 branchpoint-binding complex and other spliceosome assembly factors.

Schwer B, Chang J, Shuman S - Nucleic Acids Res. (2013)

Bottom Line: Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential.We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2.Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, NY 10065, USA and Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

ABSTRACT
Yeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

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Genetic interactions of the Msl5 PPxY100 motif. (A) Complementation of msl5Δ by the indicated MSL5 alleles was assayed by plasmid shuffle. Individual Leu+ transformants were streaked on agar medium containing FOA. Growth was scored after incubation for 7 d at 18, 25, 30 or 37°C. Lethal mutants were those that failed to form colonies at any temperature. Individual FOA-resistant colonies with viable MSL5 alleles were grown to mid-log phase in YPD broth and adjusted to equivalent A600. Serial 10-fold dilutions were spotted on YPD agar plates, which were then incubated at 25, 30, 34 and 37°C. Growth was scored as follows: (+++) colony size indistinguishable from strains bearing wild-type MSL5 at all temperatures; (++) slightly reduced colony size; (cs) pinpoint colonies at 25°C. (B) Spot testing of the growth of MSL5 WT versus Y100A in the indicated strain backgrounds at the temperatures specified.
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gkt490-F6: Genetic interactions of the Msl5 PPxY100 motif. (A) Complementation of msl5Δ by the indicated MSL5 alleles was assayed by plasmid shuffle. Individual Leu+ transformants were streaked on agar medium containing FOA. Growth was scored after incubation for 7 d at 18, 25, 30 or 37°C. Lethal mutants were those that failed to form colonies at any temperature. Individual FOA-resistant colonies with viable MSL5 alleles were grown to mid-log phase in YPD broth and adjusted to equivalent A600. Serial 10-fold dilutions were spotted on YPD agar plates, which were then incubated at 25, 30, 34 and 37°C. Growth was scored as follows: (+++) colony size indistinguishable from strains bearing wild-type MSL5 at all temperatures; (++) slightly reduced colony size; (cs) pinpoint colonies at 25°C. (B) Spot testing of the growth of MSL5 WT versus Y100A in the indicated strain backgrounds at the temperatures specified.

Mentions: To better understand how the PPxY motif functions, we tested the effects of single mutations P97A, P98A and Y100A on Msl5 activity in several genetic backgrounds for which the msl5-(P97A-P98A-Y100A) triple-mutant was lethal (e.g. mud1Δ, nam8Δ, cbc2-Y24A and tgs1Δ) or sick (mud2Δ). Initial control experiments verified that the single mutations in PPxY had no impact on complementation of msl5Δ (not shown). Complementation tests for mutational synergies revealed that the P97A and P98A alleles had no genetic interactions with mud1Δ, nam8Δ, cbc2-Y24A, tgs1Δ or mud2Δ (Figure 6A). By contrast, the Y100A allele was synthetically lethal with mud1Δ and nam8Δ and synthetic sick with mud2Δ, effectively phenocopying the synthetic defects of the 97AAxA100 triple-mutant in these backgrounds (Figure 6A). Y100A was also synthetically sick with cbc2-Y24A and tgs1Δ (Figure 6A), as evinced by smaller colony size at 34 and 37°C and cold-sensitivity (Figure 6B). These results implicate the loss of Tyr100 as principally responsible for the synthetic interactions of the Msl5 97AAxA100 mutant with proteins involved in spliceosome assembly.Figure 6.


Structure-function analysis of the 5' end of yeast U1 snRNA highlights genetic interactions with the Msl5*Mud2 branchpoint-binding complex and other spliceosome assembly factors.

Schwer B, Chang J, Shuman S - Nucleic Acids Res. (2013)

Genetic interactions of the Msl5 PPxY100 motif. (A) Complementation of msl5Δ by the indicated MSL5 alleles was assayed by plasmid shuffle. Individual Leu+ transformants were streaked on agar medium containing FOA. Growth was scored after incubation for 7 d at 18, 25, 30 or 37°C. Lethal mutants were those that failed to form colonies at any temperature. Individual FOA-resistant colonies with viable MSL5 alleles were grown to mid-log phase in YPD broth and adjusted to equivalent A600. Serial 10-fold dilutions were spotted on YPD agar plates, which were then incubated at 25, 30, 34 and 37°C. Growth was scored as follows: (+++) colony size indistinguishable from strains bearing wild-type MSL5 at all temperatures; (++) slightly reduced colony size; (cs) pinpoint colonies at 25°C. (B) Spot testing of the growth of MSL5 WT versus Y100A in the indicated strain backgrounds at the temperatures specified.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753624&req=5

gkt490-F6: Genetic interactions of the Msl5 PPxY100 motif. (A) Complementation of msl5Δ by the indicated MSL5 alleles was assayed by plasmid shuffle. Individual Leu+ transformants were streaked on agar medium containing FOA. Growth was scored after incubation for 7 d at 18, 25, 30 or 37°C. Lethal mutants were those that failed to form colonies at any temperature. Individual FOA-resistant colonies with viable MSL5 alleles were grown to mid-log phase in YPD broth and adjusted to equivalent A600. Serial 10-fold dilutions were spotted on YPD agar plates, which were then incubated at 25, 30, 34 and 37°C. Growth was scored as follows: (+++) colony size indistinguishable from strains bearing wild-type MSL5 at all temperatures; (++) slightly reduced colony size; (cs) pinpoint colonies at 25°C. (B) Spot testing of the growth of MSL5 WT versus Y100A in the indicated strain backgrounds at the temperatures specified.
Mentions: To better understand how the PPxY motif functions, we tested the effects of single mutations P97A, P98A and Y100A on Msl5 activity in several genetic backgrounds for which the msl5-(P97A-P98A-Y100A) triple-mutant was lethal (e.g. mud1Δ, nam8Δ, cbc2-Y24A and tgs1Δ) or sick (mud2Δ). Initial control experiments verified that the single mutations in PPxY had no impact on complementation of msl5Δ (not shown). Complementation tests for mutational synergies revealed that the P97A and P98A alleles had no genetic interactions with mud1Δ, nam8Δ, cbc2-Y24A, tgs1Δ or mud2Δ (Figure 6A). By contrast, the Y100A allele was synthetically lethal with mud1Δ and nam8Δ and synthetic sick with mud2Δ, effectively phenocopying the synthetic defects of the 97AAxA100 triple-mutant in these backgrounds (Figure 6A). Y100A was also synthetically sick with cbc2-Y24A and tgs1Δ (Figure 6A), as evinced by smaller colony size at 34 and 37°C and cold-sensitivity (Figure 6B). These results implicate the loss of Tyr100 as principally responsible for the synthetic interactions of the Msl5 97AAxA100 mutant with proteins involved in spliceosome assembly.Figure 6.

Bottom Line: Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential.We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2.Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Immunology Department, Weill Cornell Medical College, New York, NY 10065, USA and Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

ABSTRACT
Yeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5' splice site (5'SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5' leader between the TMG cap and the (3)ACUUAC(8) motif that anneals to the 5'SS is tolerated, there are tight constraints on the downstream spacer between (3)ACUUAC(8) and helix 1 of the U1 fold. We exploit U1 alleles with 5' extensions, variations in the (3)ACUUAC(8) motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5'SS complex with the Mud2•Msl5•BP complex.

Show MeSH
Related in: MedlinePlus