Project Grant F32GM167095

STRUCTURED LNCRNAS AS NUCLEATORS OF HETEROCHROMATIN FORMATION - GENOME INTEGRITY REQUIRES RNA-MEDIATED CONTROL OF CHROMATIN STATE, BUT THE MECHANISTIC ROLE OF RNA STRUCTURES UNDERLYING THIS PROCESS REMAINS UNCLEAR. WITH EACH CELL DIVISION, CHROMOSOME STABILITY DEPENDS ON DNA PACKING INTO TRANSCRIPTIONALLY SILENT BUNDLES CALLED HETEROCHROMATIN. EPIGENETIC MARKS DIRECT DNA CONDENSATION, AND FAILURE TO CORRECTLY DEPOSIT THESE MARKS LEADS TO CHROMOSOME MIS-SEGREGATION PATHOLOGIES, INCLUDING CANCER. THIS IS ESPECIALLY CRUCIAL AROUND CENTROMERES, THE CHROMOSOMAL ATTACHMENT POINTS. TO SPECIFY SILENCING, CELLS RECOGNIZE UNKNOWN PROPERTIES OF LONG NON-CODING RNAS (LNCRNAS) THAT ARISE FROM PERICENTROMERIC REPEATS. RNA STRUCTURE DETERMINES TRANSCRIPTS' INTERACTING PARTNERS, BUT UNTIL RECENTLY, THE IN VIVO STRUCTURES OF THESE TRANSCRIPTS HAS BEEN EXPERIMENTALLY INACCESSIBLE. THE MODEL ORGANISM FISSION YEAST (S. POMBE) PROVIDES A TRACTABLE SYSTEM TO DISSECT THIS POSSIBILITY, WITH SINGLE COPIES OF KEY GENES AND A HIGHLY CHARACTERIZED PROTEOME THAT SHARES MANY FEATURES WITH HIGHER EUKARYOTES. GROWING EVIDENCE SUGGESTS A ROLE FOR RNA INTERFERENCE (RNAI) IN CHROMATIN REGULATION IN MAMMALS, WHERE RNAI IS TYPICALLY THOUGHT TO OPERATE IN THE CYTOPLASM. HOWEVER, IN FISSION YEAST, NUCLEAR RNAI IS ESSENTIAL: A POSITIVE FEEDBACK LOOP OF SHORT INTERFERING RNAS (SIRNAS) DERIVED FROM LNCRNAS TARGET PERICENTROMERIC REPEATS TO REINFORCE HETEROCHROMATIN. THE LNCRNA REVCEN IS SUFFICIENT FOR THIS PROCESS, MAKING IT AN EXCELLENT CANDIDATE TO DISSECT RNA STRUCTURE'S ROLE IN ESTABLISHING CHROMATIN STATES. IN VITRO PROBING OF REVCEN HAS CORRELATED ITS HAIRPINS TO PRIMARY SIRNAS PRODUCED IN THE ABSENCE OF THE CANONICAL RNAI- DEPENDENT POSITIVE FEEDBACK LOOP, SUGGESTING A NOVEL PROCESSING PATHWAY. ONE CANDIDATE RNASE III-LIKE PROCESSOR, THE ESSENTIAL DROSHA HOMOLOG PAC1, COMPENSATES FOR DEFICIENCIES IN S. POMBE'S RNAI PATHWAY AND RECOGNIZES HAIRPINS, BUT ITS ACTIVITY ON PERICENTROMERIC TRANSCRIPTS IS UNKNOWN. FEW RNA STRUCTURES ARE ALLOWED TO PERSIST IN VIVO, AND THE FUNCTIONAL ROLE OF REVCEN'S PAIRING STATES REMAINS TO BE ESTABLISHED. FURTHER, THE REPERTOIRE PROTEINS THAT BIND AND RECOGNIZE REVCEN TO ACTUATE ITS FUNCTION ARE UNKNOWN. THIS PROPOSAL ADDRESSES THE HYPOTHESIS THAT STRUCTURAL FEATURES SIGNAL LOCI FOR EPIGENETIC SILENCING USING TWO COMPLEMENTARY APPROACHES. AIM 1 DEFINES THE PAIRING LANDSCAPE OF REVCEN TRANSCRIPTS IN VIVO USING MODERN CHEMICAL PROBING AND DSRNA SEQUENCING AND ESTABLISHES STRUCTURE-FUNCTION RELATIONSHIPS WITH COMPENSATORY MUTAGENESIS. BY ABLATING AND RESTORING PREDICTED HELICES AND MEASURING HETEROCHROMATIN ESTABLISHMENT, THIS STRATEGY WILL DIRECTLY LINK LNCRNA STRUCTURE TO CHROMATIN CONTROL. AIM 2 IDENTIFIES PROTEINS THAT RECOGNIZE SPECIFIC FEATURES OF LNCRNAS TO ACTUATE CHROMATIN FORMATION BY USING PARTITIONED SEGMENTS OF REVCEN AS SYNTHETIC BAITS FOR RNA PULLDOWNS FOLLOWED BY PROTEOMICS ASSAYS. PAC1'S ROLE AT THE PERICENTROMERE WILL BE DETERMINED BY BIOCHEMICAL CHARACTERIZATION ITS ACTIVITY ON REVCEN AND ACUTE DEPLETION EXPERIMENTS IN VIVO. BY UNCOVERING THE ROLE OF LNCRNA STRUCTURE AND ITS INTERPRETATION BY PROTEINS IN HETEROCHROMATIN NUCLEATION, THIS PROPOSAL WILL REVEAL A NEW AXIS IN EPIGENETIC SILENCING AND GENOME MAINTENANCE ACROSS EUKARYOTES.