Branched RNA

The only RNA molecules known to be branched are circular structures with tails known as lariats that arise during nuclear pre-mRNA splicing. Lariats accumulate within a large multicomponent particle called a spliceosome that forms upon the addition of unspliced mRNA to nuclear extracts. Recently an RNA molecule has been observed to catalyze branch formation. In this case a single intron of a yeast mitochondrial pre-mRNA participates in a self-splicing reaction that results in the accumulation of branched lariats that are processed to correctly spliced exons. An enzyme highly specific for branch removal found in the same extracts that form branches during pre-mRNA splicing can debranch RNA lariats to their linear forms without loss of nucleotides. The chemical synthesis of branched RNA has recently been achieved. High yields of sequence-specific oligonucleotides are now available for the analysis of RNA splicing by techniques dependent on branch-site recognition.     

Cis-acting elements distinct from the 5' splice site promote U1-independent pre-mRNA splicing.

We have identified a class of pre-mRNAs that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, we described pre-mRNAs that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5'' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon.     

INHIBITION OF IN VITRO PRE-mRNA SPLICING IN S. CEREVISIAE BY BRANCHED OLIGONUCLEOTIDES

A series of V- and Y-shaped nucleic acids, related to the splicing intermediates derived from S. cerevisiae actin pre-mRNA, were prepared. The effects of such branched nucleic acids (bNAs) on the efficiency of in vitro pre-mRNA splicing in yeast were studied. The exogenous bNAs each effect the efficiency of splicing, yet to different degrees, depending on the sugar composition and topology of the molecules. Y-shaped RNAs inhibited the formation of mRNA (i.e. RNA splicing) to the greatest extent.