Function and Dynamics of the LSm2-8 Protein Ring during Spliceosome Activation

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

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Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

Science ◽

10.1126/science.abc3753 ◽

2020 ◽

pp. eabc3753

Author(s):

Cole Townsend ◽

Majety N. Leelaram ◽

Dmitry E. Agafonov ◽

Olexandr Dybkov ◽

Cindy L. Will ◽

...

Keyword(s):

Active Site ◽

Rna Structure ◽

Catalytic Rna ◽

Folding Pathway ◽

Correct Order ◽

Cryo Electron Microscopy ◽

Assembly Pathway ◽

Catalytically Active ◽

Spliceosome Activation ◽

U6 Rna

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically-active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here we report the cryo-electron microscopy structures of two human pre-Bact complexes at core resolutions of 3.9-4.2 Å. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually-exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final 3D folding of the U2/U6 catalytic RNA.

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An Allosteric Network for Spliceosome Activation Revealed by High-Throughput Suppressor Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.119.301922 ◽

2019 ◽

Vol 212 (1) ◽

pp. 111-124

Author(s):

David A. Brow

Keyword(s):

Saccharomyces Cerevisiae ◽

High Throughput ◽

Spliceosome Activation ◽

Suppressor Analysis

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Spliceosome activation: U4 is the path, stem I is the goal, and Prp8 is the keeper. Let's cheer for the ATPase Brr2!

Genes & Development ◽

10.1101/gad.207514.112 ◽

2012 ◽

Vol 26 (22) ◽

pp. 2461-2467 ◽

Cited By ~ 5

Author(s):

K. H. Nielsen ◽

J. P. Staley

Keyword(s):

Spliceosome Activation

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Decision letter: Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

10.7554/elife.23533.036 ◽

2016 ◽

Keyword(s):

Small Molecule ◽

Small Molecule Inhibitor ◽

Letter Identification ◽

Early Step ◽

Molecule Inhibitor ◽

Spliceosome Activation

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Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

eLife ◽

10.7554/elife.23533 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 18

Author(s):

Anzhalika Sidarovich ◽

Cindy L Will ◽

Maria M Anokhina ◽

Javier Ceballos ◽

Sonja Sievers ◽

...

Keyword(s):

Small Molecule ◽

Intermediate Stage ◽

Mrna Splicing ◽

Molecule Inhibitor ◽

Specific Proteins ◽

Extensive Exchange ◽

Dynamics And Function ◽

Spliceosome Activation ◽

And Function ◽

Related Proteins

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.

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Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

RNA ◽

10.1261/rna.2456210 ◽

2010 ◽

Vol 16 (12) ◽

pp. 2384-2403 ◽

Cited By ~ 99

Author(s):

S. Bessonov ◽

M. Anokhina ◽

A. Krasauskas ◽

M. M. Golas ◽

B. Sander ◽

...

Keyword(s):

Morphological Changes ◽

Spliceosome Activation

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Mechanism of 5ʹ splice site transfer for human spliceosome activation

Science ◽

10.1126/science.aax3289 ◽

2019 ◽

Vol 364 (6438) ◽

pp. 362-367 ◽

Cited By ~ 30

Author(s):

Clément Charenton ◽

Max E. Wilkinson ◽

Kiyoshi Nagai

Keyword(s):

Splice Site ◽

Messenger Rna ◽

Catalytic Center ◽

U6 Snrna ◽

Dead Box ◽

Cryo Electron Microscopy ◽

Branch Point Sequence ◽

U1 Snrnp ◽

Spliceosome Activation ◽

Small Nuclear Ribonucleoproteins

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

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Crystallization and biochemical characterization of the human spliceosomal Aar2–Prp8RNaseHcomplex

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15019202 ◽

2015 ◽

Vol 71 (11) ◽

pp. 1421-1428

Author(s):

Karine Santos ◽

Marco Preussner ◽

Anna Christina Heroven ◽

Gert Weber

Keyword(s):

Biochemical Characterization ◽

Homology Modelling ◽

Molecular Replacement ◽

Noncoding Sequences ◽

Internal Loop ◽

Catalytic Activation ◽

Maturation Factor ◽

Spliceosome Activation

In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre. Thereby, Aar2p sterically excludes Brr2p, a helicase essential for the catalytic activation of the spliceosome, from Prp8p binding. At the same time, Aar2p blocks U4/U6 di-snRNA binding to Prp8p. Aar2p therefore prevents premature spliceosome activation and its functions are regulated by reversible phosphorylation. To date, little is known about the hypothetical human Aar2 (hsAar2) orthologue C20ORF4. This study identifies C20ORF4 (i) as part of the HeLa proteome by Western blotting and (ii) as a true Aar2 orthologue which binds to the RH domain (hsRH) of Prp8 and corroborates an evolutionary link between yeast and human Aar2 function. An elaborate strategy was devised to crystallize hsAar2 in complex with hsRH. The analysis of initial weakly diffracting crystals obtained byin situproteolysis and homology modelling guided the design of an hsAar2 construct in which an internal loop was replaced by three serines (hsAar2Δloop). A complex of hsAar2Δloopand hsRH crystallized in space groupC2; the crystals diffracted to 2.35 Å resolution and were suitable for structure determination by molecular-replacement approaches. The study presented here suggests a connection between Aar2 and the spliceosome in human cells and paves the way for structural studies of human Aar2.

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