RNA modifications and Prp24 coordinate Lsm2-8 binding dynamics during S. cerevisiae U6 snRNP assembly

Area of Science:

• Molecular biology
• RNA processing and splicing
• Biochemistry

Background:

• The spliceosome, a complex machine of snRNPs and proteins, removes introns from pre-mRNA in eukaryotes.
• U6 snRNP assembly is critical for splicing efficiency but its kinetics are poorly understood.
• The U6 snRNP involves U6 snRNA, Prp24, and the Lsm2-8 heteroheptamer, with a modified 3' end in yeast.

Purpose of the Study:

• To kinetically characterize the assembly pathway of the U6 snRNP.
• To investigate the roles of Prp24 protein and post-transcriptional RNA modification in U6 snRNA-Lsm2-8 complex formation.
• To understand the mechanisms governing efficient U6 snRNP reassembly for splicing.

Main Methods:

• Colocalization single-molecule spectroscopy was employed.
• Kinetic pathways of U6 snRNA association with Lsm2-8 were analyzed.
• The dependence on Prp24 and RNA modification was assessed.

Main Results:

• Lsm2-8 association with U6 snRNA is highly dependent on Prp24 without 3'-end processing.
• Processed RNAs rapidly recruit Lsm2-8, even without Prp24.
• Post-transcriptional processing enhances Lsm2-8 association, while Prp24 promotes both recruitment and retention.

Conclusions:

• Efficient U6 snRNP assembly relies on kinetic selection mechanisms.
• Lsm2-8 preferentially binds to 3'-end modified or Prp24-bound U6 snRNAs.
• This selection process likely prevents the association of Lsm2-8 with non-target RNAs, ensuring splicing fidelity.