Christopher Marshallsay

Snurportin1, an m3G‐cap‐specific nuclear import receptor with a novel domain structure

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5′‐2,2,7‐terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G‐cap but not m7G‐cap structures. Snurportin1 enhances the m3G‐capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin‐permeabilized HeLa cells, demonstrating that it functions as an snRNP‐specific nuclear import receptor. Interestingly, solely the m3G‐cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N‐terminal importin β binding (IBB) domain, essential for function, and a C‐terminal m3G‐cap‐binding region with no structural similarity to the arm repeat domain of importin α.

7-2/MRP RNAs in plant and mammalian cells : association with higher order structures in the nucleolus

Mammalian MRP (for mitochondrial RNA processing) RNA, also known as 7‐2 RNA, is a nuclear encoded small RNA which has been reported to function in two different cellular compartments: in the mitochondria and in the nucleus. The ribonucleoprotein particle which contains the 7‐2/MRP RNA, called RNase MRP, has ribonucleolytic activity and shares some structural similarity with RNase P. It has been proposed that in mitochondria, the RNase MRP is responsible for endonucleolytic cleavage of primer RNA during DNA replication. We have characterized the gene and cDNAs encoding 7‐2/MRP‐like RNA in Arabidopsis and tobacco, and found that in plants this RNA is enriched in nucleoli but is undetectable in purified mitochondria isolated from tobacco leaves or cells grown in suspension. In glycerol gradients tobacco 7‐2/MRP RNA cosediments with large approximately 80S structures possibly representing ribosomal precursors. Fractionation of HeLa cells has also revealed that 7‐2/MRP resides in the nucleolus and that most of it is associated with complexes sedimenting at approximately 80S, similar to those containing the U3 nucleolar RNA which is known to participate in pre‐rRNA processing. These results indicate that the 7‐2/MRP ribonucleoparticle may be involved in ribosome biogenesis, in both plant and mammalian cells.

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNA polymerase III.

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3′-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to α-amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.