pre-mRNA Translation Research Articles

Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA

The removal of intervening sequences from transcripts is catalyzed by the spliceosome, a multicomponent complex that assembles on the newly synthesized pre-mRNA. Pre-mRNA translation in the cytoplasm leads to the generation of aberrant proteins that are potentially harmful. Therefore, tight control to prevent undesired pre-mRNA export from the nucleus and its subsequent translation is an essential requirement for reliable gene expression. Here, we show that the natural product FR901464 (1) and its methylated derivative, spliceostatin A (2), inhibit in vitro splicing and promote pre-mRNA accumulation by binding to SF3b, a subcomplex of the U2 small nuclear ribonucleoprotein in the spliceosome. Importantly, treatment of cells with these compounds resulted in leakage of pre-mRNA to the cytoplasm, where it was translated. Knockdown of SF3b by small interfering RNA induced phenotypes similar to those seen with spliceostatin A treatment. Thus, the inhibition of pre-mRNA splicing during early steps involving SF3b allows unspliced mRNA leakage and translation.

Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm

We designed a strategy to identify splicing factors that act by preventing pre-mRNA transport into the cytoplasm. A yeast synthetic intron was inserted into a lacZ gene so that only the pre-mRNA could be translated to produce β-galactosidase activity. Deletion of either of the 5′ splice junction sequence GUAUGU and the branchpoint sequence UACUAAC resulted in a dramatic increase in pre-mRNA translation, indicating its cytoplasmic localization. In rna6 and rna9 mutant strains assayed at the nonpermissive temperature, splicing inhibition occurred simultaneously with a large increase in pre-mRNA translation. Similarly, a point mutation in U1 snRNA decreased splicing efficiency and increased pre-mRNA translation. From these results, we conclude that early acting factors, probably including U1 snRNA, and the RNA6 and RNA9 gene products, interact in vivo with the 5′ splice junction and the branchpoint sequence to commit the pre-mRNA to the splicing pathway, thereby preventing its transport to the cytoplasm.