Faculty Opinions recommendation of SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing.

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

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Mutation of the Dead-Box ATPase Prp5 Impacts Dynamics of the Reca-Like Domains and Branch Site Usage During Pre-mRNA Splicing

Biophysical Journal ◽

10.1016/j.bpj.2018.11.1170 ◽

2019 ◽

Vol 116 (3) ◽

pp. 212a

Author(s):

David H. Beier ◽

Tucker J. Carrocci ◽

Aaron A. Hoskins

Keyword(s):

Mrna Splicing ◽

Branch Site ◽

Dead Box ◽

The Dead

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Sequence Dependent Conformation and Local Geometry of the Conserved Branch Site Sequence Element d(TpApCpTpApApC), Essential for Yeast mRNA Splicing, Deduced from High Resolution1H-NMR

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.1987.10506373 ◽

1987 ◽

Vol 5 (1) ◽

pp. 35-46 ◽

Cited By ~ 2

Author(s):

Moses Lee ◽

Ding-Kwo Chang ◽

Richard T. Pon ◽

J. William Lown

Keyword(s):

Mrna Splicing ◽

Local Geometry ◽

Sequence Element ◽

Branch Site ◽

Sequence Dependent

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An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5613 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5613-5619 ◽

Cited By ~ 12

Author(s):

Y Takahashi ◽

S Urushiyama ◽

T Tani ◽

Y Ohshima

Keyword(s):

Mrna Splicing ◽

Small Nuclear Rna ◽

U2 Snrna ◽

U6 Snrna ◽

Branch Site ◽

Snrna Gene ◽

Nuclear Rna ◽

Catalytic Role ◽

Mrna Precursor ◽

Site Recognition

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

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Yeast Prp2 liberates the 5′ splice site and the branch site adenosine for catalysis of pre-mRNA splicing

RNA ◽

10.1261/rna.063115.117 ◽

2017 ◽

Vol 23 (12) ◽

pp. 1770-1779 ◽

Cited By ~ 14

Author(s):

Penghui Bao ◽

Claudia Höbartner ◽

Klaus Hartmuth ◽

Reinhard Lührmann

Keyword(s):

Splice Site ◽

Mrna Splicing ◽

Branch Site

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Variants in the 5′UTR reduce SHOX expression and contribute to SHOX haploinsufficiency

European Journal of Human Genetics ◽

10.1038/s41431-020-0676-y ◽

2020 ◽

Vol 29 (1) ◽

pp. 110-121

Author(s):

Deepak Babu ◽

Silvia Vannelli ◽

Antonella Fanelli ◽

Simona Mellone ◽

Ave Maria Baffico ◽

...

Keyword(s):

Point Mutations ◽

Mrna Splicing ◽

Luciferase Assay ◽

Transcriptional Level ◽

Coding Region ◽

Exon Trapping ◽

Branch Site ◽

Uncertain Significance ◽

Negative Effect

AbstractSHOX haploinsufficiency causes 70–90% of Léri-Weill dyschondrosteosis (LWD) and 2–10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5′UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5′UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5′UTR.

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Spatial Organization of Protein-RNA Interactions in the Branch Site-3′ Splice Site Region during pre-mRNA Splicing in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4174-4186.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4174-4186 ◽

Cited By ~ 48

Author(s):

David S. McPheeters ◽

Peggy Muhlenkamp

Keyword(s):

Protein Interactions ◽

Splice Site ◽

Spatial Organization ◽

Mrna Splicing ◽

Second Step ◽

Cross Linking ◽

Branch Site ◽

Small Nuclear Ribonucleoprotein ◽

Exon Sequence ◽

Exon Region

ABSTRACT A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3′ splice site-3′ exon region during yeast pre-mRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3′ splice site-3′ exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3′ splice site and the adjacent 3′ exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified ∼80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3′ splice site region during spliceosome assembly and catalysis.

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A Novel Type of Splicing Enhancer Regulating Adenovirus Pre-mRNA Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.20.7.2317-2325.2000 ◽

2000 ◽

Vol 20 (7) ◽

pp. 2317-2325 ◽

Cited By ~ 19

Author(s):

Oliver Mühlemann ◽

Bai-Gong Yue ◽

Svend Petersen-Mahrt ◽

Göran Akusjärvi

Keyword(s):

Virus Infection ◽

Late Phase ◽

Mrna Splicing ◽

Sequence Element ◽

Temporal Regulation ◽

Branch Site ◽

Nuclear Extracts ◽

Infected Cells ◽

Infectious Cycle ◽

Splicing Enhancer

ABSTRACT Splicing of the adenovirus IIIa pre-mRNA is subjected to a temporal regulation, such that efficient IIIa 3′ splice site usage is confined to the late phase of the infectious cycle. Here we show that IIIa pre-mRNA splicing is activated more than 200-fold in nuclear extracts prepared from late adenovirus-infected cells (Ad-NE) compared to uninfected HeLa cell nuclear extracts (HeLa-NE). In contrast, splicing of the β-globin pre-mRNA is repressed in Ad-NE. We constructed hybrid pre-mRNAs between IIIa and β-globin in order to identify the minimal IIIa sequence element conferring enhanced splicing in Ad-NE. Using this approach, we show that the IIIa branch site/pyrimidine tract functions as a Janus element: it blocks splicing in HeLa-NE and functions as a splicing enhancer in Ad-NE. Therefore, we named this sequence the IIIa virus infection-dependent splicing enhancer (3VDE). This element is essential for regulated IIIa pre-mRNA splicing in Ad-NE and sufficient to confer an enhanced splicing phenotype to the β-globin pre-mRNA in Ad-NE. We further show that the increase in IIIa splicing observed in Ad-NE is not accompanied by a similar increase in U2AF binding to the IIIa pyrimidine tract. This finding suggests that splicing activation by the 3VDE may operate without efficient U2AF interaction with the pre-mRNA. Importantly, this report represents the first description of a splicing enhancer that has evolved to function selectively in the context of a virus infection, a finding that adds a new level at which viruses may subvert the host cell RNA biosynthetic machinery to facilitate their own replication.

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