Coupling of spliceosome complexity to intron diversity

Area of Science:

• Molecular Biology
• Genetics
• Evolutionary Biology

Background:

• The spliceosome, a complex molecular machine, is essential for RNA splicing in eukaryotes.
• While many spliceosomal proteins are conserved across species, some have been lost during yeast evolution, particularly in intron-poor species like *Saccharomyces cerevisiae*.
• The functional roles of many of these lost spliceosomal proteins remain uncharacterized, especially in intron-rich organisms.

Purpose of the Study:

• To investigate the function of spliceosomal proteins conserved in fungi and humans but lost in *S. cerevisiae*.
• To determine the role of these proteins in regulating splicing efficiency and fidelity in the intron-rich yeast *Cryptococcus neoformans*.
• To identify novel protein interactions within the spliceosome that contribute to splicing accuracy.

Main Methods:

• Analysis of null mutations in a subset of uncharacterized spliceosomal factors in *Cryptococcus neoformans*.
• Assessment of splicing efficiency for introns with varying element spacing.
• Investigation of the ability of these factors to suppress cryptic/alternative splice site usage.
• Co-purification experiments to identify protein-protein interactions, including orthologs of GPATCH1 and DHX35.

Main Results:

• Null mutations in several previously uninvestigated factors impact splicing efficiency, particularly for introns with divergent element spacing.
• Most investigated factors suppress the use of weak cryptic/alternative splice sites, enhancing splicing fidelity.
• Orthologs of GPATCH1 and DHX35 function together and interact with spliceosome components, forming a conserved G patch/helicase pair.
• This pair promotes splicing fidelity, suggesting a role in preventing splicing errors.

Conclusions:

• A conserved G patch/helicase pair (GPATCH1/DHX35) plays a critical role in promoting spliceosome fidelity.
• Many spliceosomal proteins, previously lost in some yeast lineages, function to limit splicing errors in other eukaryotes.
• These error-limiting mechanisms may enable greater intron diversity and complexity in eukaryotic genomes.
• Kinetic proofreading mechanisms are proposed as a potential mode of action for these spliceosomal proteins.