Limits...
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.

Show MeSH
Effects of insertions immediately downstream of the 3ACUUAC8 sequence. The DNA sequences are shown for the 5′ ends of WT U1 and the mutant variants [+1], [+3], etc., named according to the number of nucleotides inserted between positions C8 and C9. The 3ACTTAC8 segment that pairs with the intron 5′SS is highlighted in gray. The [+5], [+10], [+20] and [+30] mutants failed to complement U1Δ in a plasmid shuffle assay and were deemed lethal. The viable U1-[+1] and U1-[+3] strains were spot-tested for growth at the indicated temperatures in parallel with WT cells as per Figure 1B, except that the plates were incubated for 5 d at 25, 20 and 18°C. The synthetic lethal interactions of the U1-[+1] and U1-[+3] mutants are indicated at the bottom of the figure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753624&req=5

gkt490-F3: Effects of insertions immediately downstream of the 3ACUUAC8 sequence. The DNA sequences are shown for the 5′ ends of WT U1 and the mutant variants [+1], [+3], etc., named according to the number of nucleotides inserted between positions C8 and C9. The 3ACTTAC8 segment that pairs with the intron 5′SS is highlighted in gray. The [+5], [+10], [+20] and [+30] mutants failed to complement U1Δ in a plasmid shuffle assay and were deemed lethal. The viable U1-[+1] and U1-[+3] strains were spot-tested for growth at the indicated temperatures in parallel with WT cells as per Figure 1B, except that the plates were incubated for 5 d at 25, 20 and 18°C. The synthetic lethal interactions of the U1-[+1] and U1-[+3] mutants are indicated at the bottom of the figure.

Mentions: The 5′ AUACUUACCU10 single-stranded segment of U1 snRNA precedes the folded U1 RNA tertiary structure that initiates at nucleotide U11 (i.e. a four-helix junction depicted in Figure 4A). The 8CCU10 segment connecting the 5′SS complementary motif to helix 1 interacts with the U1-C subunit in the U1 snRNP (4,8). Here, we queried the importance of the spacing between the 5′SS complementary motif and helix 1 by introducing insertions of 1–30 nt between C8 and C9 (Figure 3). The insertion alleles were tested for bioactivity by plasmid shuffle in the U1Δ strain. The [+1] and [+3] insert strains were viable at 18–37°C, albeit slower growing than WT U1 cells at low temperatures (Figure 3). By contrast, insertions of ≥5 nt between C8 and C9 were uniformly lethal (Figure 3). We performed primer extension analysis on RNA isolated from yeast cells with a wild-type chromosomal U1 gene that had been transformed with CEN plasmids bearing the series of U1 insertion alleles (Supplementary Figure S2). Denaturing PAGE analysis of the 5′ 32P-labeled primer extension products revealed that the 5′ ends of the plasmid-encoded [+1], [+3], [+5], [+10], [+20] and [+30] U1 snRNAs were shifted incrementally upstream compared with the endogenous wild-type U1 snRNA. The levels of radiolabeled cDNAs derived from the U1 snRNA insertion mutants were at least as high as the cDNA corresponding to endogenous wild-type U1 snRNA (Supplementary Figure S2), signifying that (i) the steady-state levels of the U1 snRNA were not affected by the inserted sequences and (ii) the lethality of the [+5], [+10], [+20] and [+30] U1 alleles was not attributable to a failure the produce the mutant snRNAs. We surmise that there are tight constraints on the length (and/or sequence) of the linker segment that interacts with U1-C. This theme was underscored as we systematically tested the viable [+1] and [+3] insert alleles for mutational synergies, which revealed that the [+1] and [+3] insertions were lethal in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ. Thus, even a single extra nucleotide sufficed to render U1 snRNA function dependent on otherwise inessential components of the U1 snRNP or the early spliceosome.Figure 3.


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)

Effects of insertions immediately downstream of the 3ACUUAC8 sequence. The DNA sequences are shown for the 5′ ends of WT U1 and the mutant variants [+1], [+3], etc., named according to the number of nucleotides inserted between positions C8 and C9. The 3ACTTAC8 segment that pairs with the intron 5′SS is highlighted in gray. The [+5], [+10], [+20] and [+30] mutants failed to complement U1Δ in a plasmid shuffle assay and were deemed lethal. The viable U1-[+1] and U1-[+3] strains were spot-tested for growth at the indicated temperatures in parallel with WT cells as per Figure 1B, except that the plates were incubated for 5 d at 25, 20 and 18°C. The synthetic lethal interactions of the U1-[+1] and U1-[+3] mutants are indicated at the bottom of the figure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt490-F3: Effects of insertions immediately downstream of the 3ACUUAC8 sequence. The DNA sequences are shown for the 5′ ends of WT U1 and the mutant variants [+1], [+3], etc., named according to the number of nucleotides inserted between positions C8 and C9. The 3ACTTAC8 segment that pairs with the intron 5′SS is highlighted in gray. The [+5], [+10], [+20] and [+30] mutants failed to complement U1Δ in a plasmid shuffle assay and were deemed lethal. The viable U1-[+1] and U1-[+3] strains were spot-tested for growth at the indicated temperatures in parallel with WT cells as per Figure 1B, except that the plates were incubated for 5 d at 25, 20 and 18°C. The synthetic lethal interactions of the U1-[+1] and U1-[+3] mutants are indicated at the bottom of the figure.
Mentions: The 5′ AUACUUACCU10 single-stranded segment of U1 snRNA precedes the folded U1 RNA tertiary structure that initiates at nucleotide U11 (i.e. a four-helix junction depicted in Figure 4A). The 8CCU10 segment connecting the 5′SS complementary motif to helix 1 interacts with the U1-C subunit in the U1 snRNP (4,8). Here, we queried the importance of the spacing between the 5′SS complementary motif and helix 1 by introducing insertions of 1–30 nt between C8 and C9 (Figure 3). The insertion alleles were tested for bioactivity by plasmid shuffle in the U1Δ strain. The [+1] and [+3] insert strains were viable at 18–37°C, albeit slower growing than WT U1 cells at low temperatures (Figure 3). By contrast, insertions of ≥5 nt between C8 and C9 were uniformly lethal (Figure 3). We performed primer extension analysis on RNA isolated from yeast cells with a wild-type chromosomal U1 gene that had been transformed with CEN plasmids bearing the series of U1 insertion alleles (Supplementary Figure S2). Denaturing PAGE analysis of the 5′ 32P-labeled primer extension products revealed that the 5′ ends of the plasmid-encoded [+1], [+3], [+5], [+10], [+20] and [+30] U1 snRNAs were shifted incrementally upstream compared with the endogenous wild-type U1 snRNA. The levels of radiolabeled cDNAs derived from the U1 snRNA insertion mutants were at least as high as the cDNA corresponding to endogenous wild-type U1 snRNA (Supplementary Figure S2), signifying that (i) the steady-state levels of the U1 snRNA were not affected by the inserted sequences and (ii) the lethality of the [+5], [+10], [+20] and [+30] U1 alleles was not attributable to a failure the produce the mutant snRNAs. We surmise that there are tight constraints on the length (and/or sequence) of the linker segment that interacts with U1-C. This theme was underscored as we systematically tested the viable [+1] and [+3] insert alleles for mutational synergies, which revealed that the [+1] and [+3] insertions were lethal in combination with tgs1Δ, cbc2-Y24A, nam8Δ, mud1Δ, swt21Δ and mud2Δ. Thus, even a single extra nucleotide sufficed to render U1 snRNA function dependent on otherwise inessential components of the U1 snRNP or the early spliceosome.Figure 3.

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