A search for cellular components that interact with the U14 small nucleolar RNA of yeast

摘要

U14 is an evolutionary conserved small nucleolar RNA required for 18S ribosomal RNA production in the yeast Saccharomyces cerevisiae. This study attempts to identify cellular components that interact with U14 RNA with the aim of gaining insight into U14 function and identifying strategies for characterizing this activity. The objectives include: (1) development of a hypothetical secondary folding model for U14 RNA; (2) biochemical characterization of the U14 small nuclear ribonucleoprotein particle (snRNP); (3) identification of U14 binding proteins through biochemical and genetic approaches, and; (4) a genetic test to determine if U14 interacts with 18S RNA through an essential, complementary segment in U14 RNA, i.e. domain 18S-A.;Two U14 consensus secondary structures were established by phylogenetic folding analysis. These structures are supported by a variety of genetic observations, but are only partially consistent with biochemical probing data developed by others. Additional research is required to determine the actual folding properties of U14 in vivo.;Biochemical characterization showed that natural U14 RNA does not contain a trimethylguanosine (TMG) cap, while U14 synthesized from the GAL1 promoter does. Natural U14 occurs in two classes of RNP complexes: (i) a 10S RNP presumed to be a free snRNP and, (ii) a polydisperse, larger complex containing fibrillarin. The direct association of fibrillarin with in vitro synthesized U14 was not detected using a variety of assays. Analysis of the higher order structures sedimenting at approximately 90S revealed the presence of over one dozen snRNAs, two-thirds of which appear to be associated with fibrillarin.;Several extragenic suppressor mutations were collected that relieve a lethal mutation in the box C sequence. One of these was identified as a putative sugar transport protein. It seems unlikely that this protein is directly related to U14 function per se, but may play a role in the regulation of U14 activity, possibly by influencing proteins that interact with U14 RNA or DNA.;Mutations in a domain A-related sequence of 18S rDNA resulted in under-accumulation of 18S RNA, demonstrating the importance of this region for 18S RNA stability. This effect was not influenced by complementary changes in domain A of U14 RNA.;The significance of these results are discussed in the context of U14 function and ribosome biogenesis.