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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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U1 snRNAs with extended 5′ ends are functional. (A) The DNA sequences are shown for the 5′ ends of wild-type U1 (WT) and the mutant variants +5, +10 and so forth, named according to the number of nucleotides inserted upstream of the 3ACTTAC8 segment (highlighted in gray) that pairs with the intron 5′SS. (B) The growth phenotypes of U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were assessed as follows. Liquid cultures were grown to mid-log phase at 30°C and adjusted to the same A600. Aliquots (3 µl) of serial 10-fold dilutions of cells were spotted to YPD agar. The plates were incubated at the indicated temperatures and photographed after 2 d (30, 34 and 37°C), 3 d (25°C) or 4 d (20°C). (C) Primer extension analyses with 32P-labeled primers complementary to U1 snRNA (nt 161–182 ) and U2 snRNA (nt 140–160 ) was performed using as template total cellular RNA isolated from the indicated U1Δ p(CEN LEU2 U1) strains. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography. The sizes (nt) of 32P-labeled marker DNAs that were analyzed in parallel are indicated at left.
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gkt490-F1: U1 snRNAs with extended 5′ ends are functional. (A) The DNA sequences are shown for the 5′ ends of wild-type U1 (WT) and the mutant variants +5, +10 and so forth, named according to the number of nucleotides inserted upstream of the 3ACTTAC8 segment (highlighted in gray) that pairs with the intron 5′SS. (B) The growth phenotypes of U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were assessed as follows. Liquid cultures were grown to mid-log phase at 30°C and adjusted to the same A600. Aliquots (3 µl) of serial 10-fold dilutions of cells were spotted to YPD agar. The plates were incubated at the indicated temperatures and photographed after 2 d (30, 34 and 37°C), 3 d (25°C) or 4 d (20°C). (C) Primer extension analyses with 32P-labeled primers complementary to U1 snRNA (nt 161–182 ) and U2 snRNA (nt 140–160 ) was performed using as template total cellular RNA isolated from the indicated U1Δ p(CEN LEU2 U1) strains. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography. The sizes (nt) of 32P-labeled marker DNAs that were analyzed in parallel are indicated at left.

Mentions: To test this idea, we extended the 5′ sequence of the U1 gene by 5, 10, 15, 20, 25 or 30 nt, as depicted in Figure 1A. The wild-type (WT) and 5′-extended U1 alleles under the control of the native U1 promoter were placed on CEN LEU2 plasmids and tested for function in vivo by plasmid shuffle in a yeast strain deleted at the chromosomal U1 locus but bearing a WT U1 gene on a CEN URA3 plasmid. All of the 5′-extended U1 alleles supported growth of yeast U1Δ cells on medium containing FOA, a drug that selects against the CEN URA3 U1 plasmid. The U1 +5, +10, +15, +20, +25 and +30 strains grew as well as U1 WT cells at 20, 25, 30, 34 and 37°C, as gauged by spotting serial dilutions of the respective strains on YPD agar (Figure 1B). To assess whether the newly inserted sequences at the 5′ end of the U1 gene were transcribed into the U1 snRNA, we performed primer extension analysis on total RNA isolated from yeast cells bearing the WT and 5′-extended U1 alleles (Figure 1C). A 5′ 32P-labeled DNA oligonucleotide complementary to yeast U1 snRNA was extended by reverse transcriptase to yield a discrete cDNA corresponding in size to the distance (in nucleotides) from the primer 5′ end to the 5′ end of the U1 snRNA. A second 5′ 32P-labeled DNA oligonucleotide complementary to U2 snRNA was included in the reverse transcription reactions as a control. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the primer extension products revealed that the 5′ ends of the U1 snRNAs were shifted serially ‘upstream’ by 5-nt intervals, as expected from the U1 DNA sequences. Thus, the DNA additions did not alter the site of transcription initiation directed by the 5′-flanking U1 gene promoter. The amounts of U1 cDNA synthesized from total RNAs derived from the WT and 5′-extended U1 yeast strains were similar, signifying that the steady-state levels of U1 snRNA were not affected by the 5′ leader sequences. The 5′ ends and steady-state levels of U2 snRNAs were unaltered by the U1 mutations.Figure 1.


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)

U1 snRNAs with extended 5′ ends are functional. (A) The DNA sequences are shown for the 5′ ends of wild-type U1 (WT) and the mutant variants +5, +10 and so forth, named according to the number of nucleotides inserted upstream of the 3ACTTAC8 segment (highlighted in gray) that pairs with the intron 5′SS. (B) The growth phenotypes of U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were assessed as follows. Liquid cultures were grown to mid-log phase at 30°C and adjusted to the same A600. Aliquots (3 µl) of serial 10-fold dilutions of cells were spotted to YPD agar. The plates were incubated at the indicated temperatures and photographed after 2 d (30, 34 and 37°C), 3 d (25°C) or 4 d (20°C). (C) Primer extension analyses with 32P-labeled primers complementary to U1 snRNA (nt 161–182 ) and U2 snRNA (nt 140–160 ) was performed using as template total cellular RNA isolated from the indicated U1Δ p(CEN LEU2 U1) strains. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography. The sizes (nt) of 32P-labeled marker DNAs that were analyzed in parallel are indicated at left.
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Related In: Results  -  Collection

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gkt490-F1: U1 snRNAs with extended 5′ ends are functional. (A) The DNA sequences are shown for the 5′ ends of wild-type U1 (WT) and the mutant variants +5, +10 and so forth, named according to the number of nucleotides inserted upstream of the 3ACTTAC8 segment (highlighted in gray) that pairs with the intron 5′SS. (B) The growth phenotypes of U1Δ p(CEN LEU2 U1) cells bearing the indicated U1 alleles were assessed as follows. Liquid cultures were grown to mid-log phase at 30°C and adjusted to the same A600. Aliquots (3 µl) of serial 10-fold dilutions of cells were spotted to YPD agar. The plates were incubated at the indicated temperatures and photographed after 2 d (30, 34 and 37°C), 3 d (25°C) or 4 d (20°C). (C) Primer extension analyses with 32P-labeled primers complementary to U1 snRNA (nt 161–182 ) and U2 snRNA (nt 140–160 ) was performed using as template total cellular RNA isolated from the indicated U1Δ p(CEN LEU2 U1) strains. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography. The sizes (nt) of 32P-labeled marker DNAs that were analyzed in parallel are indicated at left.
Mentions: To test this idea, we extended the 5′ sequence of the U1 gene by 5, 10, 15, 20, 25 or 30 nt, as depicted in Figure 1A. The wild-type (WT) and 5′-extended U1 alleles under the control of the native U1 promoter were placed on CEN LEU2 plasmids and tested for function in vivo by plasmid shuffle in a yeast strain deleted at the chromosomal U1 locus but bearing a WT U1 gene on a CEN URA3 plasmid. All of the 5′-extended U1 alleles supported growth of yeast U1Δ cells on medium containing FOA, a drug that selects against the CEN URA3 U1 plasmid. The U1 +5, +10, +15, +20, +25 and +30 strains grew as well as U1 WT cells at 20, 25, 30, 34 and 37°C, as gauged by spotting serial dilutions of the respective strains on YPD agar (Figure 1B). To assess whether the newly inserted sequences at the 5′ end of the U1 gene were transcribed into the U1 snRNA, we performed primer extension analysis on total RNA isolated from yeast cells bearing the WT and 5′-extended U1 alleles (Figure 1C). A 5′ 32P-labeled DNA oligonucleotide complementary to yeast U1 snRNA was extended by reverse transcriptase to yield a discrete cDNA corresponding in size to the distance (in nucleotides) from the primer 5′ end to the 5′ end of the U1 snRNA. A second 5′ 32P-labeled DNA oligonucleotide complementary to U2 snRNA was included in the reverse transcription reactions as a control. Denaturing polyacrylamide gel electrophoresis (PAGE) analysis of the primer extension products revealed that the 5′ ends of the U1 snRNAs were shifted serially ‘upstream’ by 5-nt intervals, as expected from the U1 DNA sequences. Thus, the DNA additions did not alter the site of transcription initiation directed by the 5′-flanking U1 gene promoter. The amounts of U1 cDNA synthesized from total RNAs derived from the WT and 5′-extended U1 yeast strains were similar, signifying that the steady-state levels of U1 snRNA were not affected by the 5′ leader sequences. The 5′ ends and steady-state levels of U2 snRNAs were unaltered by the U1 mutations.Figure 1.

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