Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3′-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module. The composite double-stranded/single-stranded RNA-binding region assembles cooperatively with Snu13 and Prp31 on U4/U6 di-snRNAs and inhibits Brr2-mediated U4/U6 di-snRNA unwinding in vitro. RNP-disrupting mutations in Prp3 lead to U4/U6•U5 tri-snRNP assembly and splicing defects in vivo. Our results reveal how Prp3 acts as an important bridge between U4/U6 and U5 in the tri-snRNP and comparison with a Prp24-U6 snRNA recycling complex suggests how Prp3 may be involved in U4/U6 reassembly after splicing.
Proteins are built following instructions contained within the DNA of gene sequences. This genetic information is copied into short-lived molecules, called messenger RNAs (or mRNAs), which move away from the DNA and are then decoded by the molecular machines that build proteins. However, mRNA sequences often have to be edited before they are used. Another molecular machine, called a spliceosome, carries out some of this editing.
A spliceosome is formed from a number of smaller subunits, including three RNA-protein particles that each contain one RNA molecule (called U1, U2 and U5), and one particle that contains two RNA molecules (called U4 and U6). These subunits must assemble around an unedited mRNA in a particular order so that the spliceosome can work correctly. Once the mRNA has been edited, and the spliceosome has performed its job, these complexes need to disassemble so that they are ready to be reassembled around a new mRNA molecule. A protein called Prp3 is known to be involved in these assembly, disassembly and reassembly steps. However, it is unclear how this protein performs these activities.
Liu et al. have now used structural biology and biochemical techniques to determine the three-dimensional structure of Prp3, and have shown that this protein has a “two-part” binding site that binds to the RNA molecules in the U4/U6 subunit of the spliceosome. Further analyses revealed that one of these features is only found in Prp3 and not in other types of RNA-binding proteins.
Together with previous work, Liu et al. also reveal that Prp3 can serve as a ‘bridge’ between the U4/U6 and U5 subunits of the spliceosome, and suggest how these features allow the two subunits to group together before they are incorporated into a spliceosome.
Notably, certain mutations in the gene for the Prp3 protein lead to a human eye disease called retinitis pigmentosa. In the future it will be important to investigate if the above activities are affected in the mutant variants of the Prp3 protein.