HUMAN SMALL NUCLEAR RNAS WITH NOVEL NUCLEOTIDE SEQUENCES

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We have synthesized and Cloned novel cDNA probes for three new human small nucleolar RNAs of unique nucleotide sequences, that will be named R, S and T. RNAs R, S and T appear to be true small RNA species, as opposed to breakdown products of large RNAs, because their cDNAs hybridize to a small RNA, but not to large RNA, in agarose gel Northern blots. Our long-term objective is to determine the function of RNAs R, S and T. It is reasonable to suspect that these small RNAs are involved in some aspect of ribosome production, because of their nucleolar location. Sequence complementarities between RNAs R-T and pre-rRNA are compatible with possible roles in pre-rRNA processing. This proposal focuses on testing the hypothesis that these small RNAs may function in the pathway of ribosome biogenesis. Our first goal is to determine the sequence of the -42 nucleotides located at the 5' end of RNA S. Some molecules of RNAs T and S are psoralen-crosslinked in vivo to large nucleolar RNA. This large nucleolar RNA consists of two main bands when hybridized with the S DNA probe: one migrates near 28S rRNA and the other is larger. Our second goal is to find out whether a small percentage of the molecules of RNAs R-T: a) behave as if specifically associated with pre-rRNA RNP particles or pre-rRNA and b) can be specifically psoralen-crosslinked in vivo to pre-rRNA, and, if so, to map approximately the location of the intermolecular crosslink in pre-rRNA. The third goal is the identification of the probes or system necessary to specifically lower the level of available, intact molecules of RNAs R-T. One antisense oligodeoxynucleotide targets the specific degradation of the RNA T present in a nucleolar extract. The first choice is to locate the accessible regions of RNAs R-T in nucleolar extracts and whole cells, using antisense oligodeoxynucleotide-targeted degradation. Alternative approaches are discussed. Nucleotide sequences that are highly conserved through evolution tend to be functionally important. Then, in our fourth goal a phylogenetic comparison of the nucleotide sequences of RNAs R-T should identify the conserved sequences. The fifth goal is to test the effect of the induced decrease of the level of available, intact molecules of RNAs R, S and T on the pathway of ribosome formation in whole cells or cell-free systems. There are important questions about the proteins with which these small RNAs may associate, and about the 5' ends of these small RNAs, using as probes currently available antibodies. We wish to ask those questions as this project progresses, when time allows it.