Vertebrate cells employ a litany of non-coding RNAs (ncRNAs) to orchestrate and regulate the synthesis of proteins. In addition to the transfer RNAs and ribosomal RNAs that decode messenger RNAs in the cytoplasm, ncRNAs are involved in the earliest steps of gene expression in the nucleus: long ncRNAs modulate chromatin structure and thus transcription, while two sets of small nuclear RNAs (snRNAs) are the building blocks of the major and minor spliceosomes that remove both constitutively- and alternatively-spliced introns from pre- mRNAs. Even the 32-end formation of histone mRNAs requires the U7 snRNA. These essential ncRNAs themselves undergo covalent modification directed by yet other classes of ncRNAs during their biogenesis in the nucleus: small nucleolar RNAs (snoRNAs) and small Cajal body RNAs (scaRNAs) guide the specific introduction of 22-O-methyl and pseudoU residues into rRNAs and snRNAs, respectively. Once exported to the cytoplasm, mRNA translation and stability are regulated by microRNAs, the newest class of ncRNAs, which undergo regulated processing steps in both the nucleus and the cytoplasm during their biogenesis. All of these ncRNAs act in conjunction with bound proteins, forming ncRNPs, to execute their functions. The proposed research will continue to discover novel ncRNP functions and protein interactions that underlie the biogenesis and stability of ncRNAs, particularly in the vertebrate cell nucleus. Specifically, a novel chaperone function for a scaRNA in assembling the U2 snRNP will be investigated. Our discovery that the core RNA helicase component (eIF4AIII) of the exon junction complex has a second conserved role in ribosomal RNA biogenesis will be confirmed. The probable existence of additional eIF4A/eIF4G-like pairs will be explored and their contributions to critical steps in gene expression assigned. Phosphoproteomics and single-molecule approaches will complement functional studies of the core components of the Microprocessor-Drosha and DGCR8-to uncover the molecular basis of regulation of pri-microRNA processing in the nucleus of tumorigenic versus non-tumorigenic cells. RNA elements that confer stability on nuclear long noncoding RNAs will be identified bioinformatically and characterized both biochemically and structurally. Nucleases participating in rapid RNA decay in the nucleus will be identified, and their possible localization in nuclear bodies will be investigated using single-molecule live-cell imaging techniques. Together, this understanding will contribute to the development of therapeutic approaches to combat multiple genetic diseases that derive from faulty RNA processing and surveillance, as well as cancer.

Public Health Relevance

Some RNA molecules do not serve as messages for making proteins but nonetheless play essential roles in the production of the cell's repertoire of proteins. Recently, additional kinds and novel functions of such noncoding RNAs have been recognized, underscoring their importance in regulating the production of cellular proteins. Our exploration of the contributions of noncoding RNAs will provide novel insights into potential targets and therapeutic mechanisms for combating a multitude of devastating genetic diseases including muscular dystrophy, spinal muscular atrophy and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026154-44
Application #
8664862
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Bender, Michael T
Project Start
1979-05-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
44
Fiscal Year
2014
Total Cost
$283,050
Indirect Cost
$113,050
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Brown, Jessica A; Bulkley, David; Wang, Jimin et al. (2014) Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix. Nat Struct Mol Biol 21:633-40
Cech, Thomas R; Steitz, Joan A (2014) The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157:77-94
Xie, Mingyi; Steitz, Joan A (2014) Versatile microRNA biogenesis in animals and their viruses. RNA Biol 11:673-81
Riley, Kasandra J; Steitz, Joan A (2013) The "Observer Effect" in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601-4
Herbert, Kristina M; Pimienta, Genaro; DeGregorio, Suzanne J et al. (2013) Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile. Cell Rep 5:1070-81
Alexandrov, Andrei; Colognori, David; Steitz, Joan A (2011) Human eIF4AIII interacts with an eIF4G-like partner, NOM1, revealing an evolutionarily conserved function outside the exon junction complex. Genes Dev 25:1078-90
Mortensen, Richard D; Serra, Maria; Steitz, Joan A et al. (2011) Posttranscriptional activation of gene expression in Xenopus laevis oocytes by microRNA-protein complexes (microRNPs). Proc Natl Acad Sci U S A 108:8281-6
Pawlicki, Jan M; Steitz, Joan A (2010) Nuclear networking fashions pre-messenger RNA and primary microRNA transcripts for function. Trends Cell Biol 20:52-61
Borah, Sumit; Wong, Anthony C; Steitz, Joan A (2009) Drosophila hnRNP A1 homologs Hrp36/Hrp38 enhance U2-type versus U12-type splicing to regulate alternative splicing of the prospero twintron. Proc Natl Acad Sci U S A 106:2577-82
Tycowski, Kazimierz T; Shu, Mei-Di; Kukoyi, Abiodun et al. (2009) A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol Cell 34:47-57

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