Project Grant F32GM167162

ELUCIDATING THE MECHANISMS OF FMRP-MEDIATED TRANSLATION REPRESSION THROUGH STRUCTURAL AND BIOCHEMICAL ANALYSES OF FMRP-BOUND MRNPS - FRAGILE X SYNDROME (FXS) IS THE LEADING SINGLE-GENE CAUSE OF INTELLECTUAL DISABILITY AND AUTISM AND IS CAUSED BY THE LOSS OF EXPRESSION OF THE FRAGILE X PROTEIN (FMRP) DUE TO TRANSCRIPTIONAL SILENCING OF THE FMR1 GENE, WHICH ENCODES FMRP. FMRP IS AN RNA-BINDING PROTEIN THAT BINDS AN ESTIMATED 6000 CELLULAR MESSENGER RNAS (MRNAS) TO FORM MESSENGER RIBONUCLEOPROTEIN COMPLEXES (MRNPS). FMRP-BOUND MRNPS ARE SEQUESTERED INTO A TRANSLATIONALLY SILENCED STATE AND PROTECTED FROM DEGRADATION BY DEADENYLATION. THEREFORE, THE LOSS OF FMRP LEADS TO INCREASED TRANSLATION AND SUBSEQUENT DECAY OF ITS TARGET MRNAS, RESULTING IN DYSREGULATED PROTEIN PRODUCTION. NOTABLY, THE MECHANISMS BY WHICH FMRP REPRESSES TRANSLATION HAVE YET TO BE FULLY ELUCIDATED. DATA FROM THE MAQUAT LAB INDICATE THAT FMRP REPRESSES THE TRANSLATION OF ITS TARGET MRNAS AT TRANSLATION INITIATION RATHER THAN AT TRANSLATION ELONGATION, THE LATTER OF WHICH IS SUGGESTED BY PREVIOUS WORK IN THE FIELD. WE HAVE SHOWN THAT REPRESSION TYPIFIES BOTH NEWLY MADE FMRP-BOUND MRNPS, DEFINED AS THOSE HARBORING THE CAP- BINDING PROTEIN HETERODIMER CBP80-CBP20 (CBC) AT THE MRNA 5'-CAP, AND THEIR REMODELED STEADY STATE PRODUCTS, DEFINED AS THOSE HARBORING EIF4E AT THE MRNA 5'-CAP. TO DATE, WE HAVE SHOWN THAT FMRP BINDS ITS TARGET EIF4E-BOUND MRNPS THROUGH DIRECT INTERACTIONS WITH (I) THE TRANSCRIBED BODY OF THE MRNA, (II) EIF4E AT THE 5'-CAP AND (III) POLY(A)-BINDING PROTEIN C1 (PABPC1) AT THE MRNA 3'-POLY(A) TAIL. WE HAVE ALSO SHOWN THAT FMRP DOES NOT BIND THE CBC DIRECTLY. OUR DATA INDICATE THAT THE MECHANISMS BY WHICH FMRP REPRESSES TRANSLATION (I) DIFFER FROM EXISTING VIEWS AND (II) DIFFER FOR EIF4E-BOUND FMRP-TARGET MRNPS RELATIVE TO CBC- BOUND FMRP-TARGET MRNPS. IN THIS PROPOSAL, I AIM TO CHARACTERIZE FMRP-BOUND MRNPS THROUGHOUT THEIR LIFE CYCLE TO ELUCIDATE THE MECHANISMS OF FMRP-MEDIATED TRANSLATION REPRESSION. IN AIM 1, I WILL UTILIZE CRYOGENIC ELECTRON MICROSCOPY (CRYO-EM) FOR THE ANALYSES OF THREE IN VITRO-FORMED COMPLEXES TO IDENTIFY THEIR INTERACTION INTERFACES. I WILL THEN USE LYSATES OF CELLS EXPRESSING THE APPROPRIATE PROTEIN VARIANTS AND BIOCHEMICAL ANALYSES TO VALIDATE THE IDENTIFIED INTERACTING RESIDUES. IN AIM 2, I WILL DETERMINE HOW MECHANISMS OF FMRP-MEDIATED TRANSLATION REPRESSION DIFFER BETWEEN NEWLY MADE CBC-BOUND MRNPS AND STEADY STATE EIF4E-BOUND MRNPS. I WILL ISOLATE EACH TYPE OF FMRP-BOUND MRNP USING CELL LYSATES OF ENGINEERED CELLS AND SEQUENTIAL PURIFICATION STEPS TO DEFINE CONSTITUENTS OF EACH USING LIQUID-CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY. AFTER DATA IS OBTAINED, I WILL THEN GENERATE AUXIN-INDUCIBLE DEGRON (AID) CELL LINES TO TEST WHICH OF THE IDENTIFIED CONSTITUENTS IS REQUIRED FOR MRNP INTEGRITY AND FMRP-MEDIATED TRANSLATION REPRESSION. RESULTS WILL PROVIDE INSIGHT INTO THE POTENTIAL MECHANISMS BY WHICH TRANSLATION INITIATION CAN BE REGULATED AND HIGHLIGHT MECHANISMS BY WHICH NEWLY MADE MRNPS AND STEADY STATE MRNPS ARE DIFFERENTIALLY REGULATED. THIS WORK IS EXPECTED TO ADVANCE OUR UNDERSTANDING OF TRANSLATION REGULATION AND HOW A LOSS OF REGULATION CAN LEAD TO NEURODEVELOPMENTAL PATHOLOGY.