Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene.

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.     

The yeast RNA gene products are essential for mRNA splicing in vitro

The yeast rna mutations (rna2-rna11) are a set of temperature-sensitive mutations that result in the accumulation of intron-containing mRNA precursors at the restrictive temperature. We have used the yeast in vitro splicing system to investigate the role of products of the RNA genes in mRNA splicing. We have tested the heat lability of the in vitro mRNA splicing reaction in extracts isolated from mutant and wild-type cells. Extracts isolated from seven of the nine rna mutants demonstrated heat lability in this assay, while most wild-type extracts were stable under the conditions utilized. We have also demonstrated that heat inactivation usually results in the specific loss of an exchangeable component by showing that most combinations of heat-inactivated extracts from different mutants complement one another. In three cases (rna2, rna5, and rna11), the linkage of the in vitro defect to the rna mutations was ascertained by a combination of reversion, tetrad, and in vitro complementation analyses. Furthermore, each heat-inactivated extract was capable of complementation by at least one fraction of the wild-type splicing system. Thus many of the RNA genes are likely to code for products directly involved in and essential for mRNA splicing.