Structural insights into branch site proofreading by human spliceosome.

Area of Science:

• Molecular Biology
• Structural Biology
• RNA Splicing

Background:

• Prespliceosome assembly requires accurate branch site (BS) selection by U2 small nuclear ribonucleoprotein (snRNP).
• The RNA helicase PRP5 is known to proofread BS selection, but its mechanism is not fully understood.
• Understanding this process is crucial for comprehending gene expression regulation and associated diseases.

Purpose of the Study:

• To elucidate the atomic mechanisms by which PRP5 proofreads branch site selection during prespliceosome assembly.
• To determine the structural basis of U2 snRNP and pre-mRNA interactions in early splicing complexes.
• To investigate the role of specific splicing factors and the impact of cancer-associated mutations.

Main Methods:

• Determining the atomic structures of key intermediate complexes using cryo-electron microscopy.
• Analyzing protein-RNA interactions within the 17S U2 snRNP and pre-A complex.
• Investigating the functional consequences of mutations in SF3B1 on PRP5 binding and BS proofreading.

Main Results:

• Atomic structures of the 17S U2 snRNP and a cross-exon pre-A complex were determined.
• PRP5 is anchored to U2 snRNP via an acidic loop interacting with SF3B1, while its helicase domain associates with U2 snRNA.
• The BS-interacting stem-loop (BSL) of U2 snRNA is initially shielded, and later forms a duplex with the BS in the pre-A complex, stabilized by SF1, DNAJC8, and SF3A2.
• Cancer-derived SF3B1 mutations disrupt PRP5 association, impairing BS proofreading.

Conclusions:

• PRP5 proofreading of BS selection involves dynamic structural rearrangements and specific interactions within the prespliceosome.
• The structures provide a mechanistic basis for PRP5's role in ensuring splicing fidelity.
• SF3B1 mutations linked to cancer compromise this crucial proofreading step, highlighting its importance in disease pathogenesis.