PhD Proposal by Luke Knudson

Event Details
  • Date/Time:
    • Tuesday February 2, 2021 - Wednesday February 3, 2021
      2:00 pm - 2:59 pm
  • Location: Atlanta, GA
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  • URL: Zoom
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Summaries

Summary Sentence: MUSCLEBLIND-LIKE RNA BINDING PROTEIN MEDIATED mRNA TRANSPORT BY SELECTIVE KINESINS NEURONS AND DYSREGULATION IN MYTONIC DYSTROPHY

Full Summary: No summary paragraph submitted.

Luke Knudson

BME PhD Thesis Proposal Presentation

 

Date: 02/03/2021

 

Time: 2:00p - 3:00p

Location: https://emory.zoom.us/j/97022856590?pwd=S3J1dVlXdHN2SWZza2J5alJXaTFjZz09&from=addon 

 

Meeting ID: 970 2285 6590 

Passcode: 437561

 

 

Faculty Advisor: 

Dr. Gary Bassell

 

Committee Members:  

Dr. Philip Santangelo

Dr. Karmella Haynes

Dr. Dorothy Lerit

Dr. Anita Corbett 

 

Title: MUSCLEBLIND-LIKE RNA BINDING PROTEIN MEDIATED mRNA TRANSPORT BY SELECTIVE KINESINS NEURONS AND DYSREGULATION IN MYTONIC DYSTROPHY

 

Abstract: 

Myotonic dystrophy (dystrophia myotonica, DM) is an autosomal dominant genetic disease, with a diagnosed prevalence of 1:8000 people worldwide, that affects multiple tissues of the body, including skeletal muscle, heart, and the central nervous system (CNS). Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats in the 3' UTR of dystrophia myotonica protein kinase (DMPK) gene. Studies in mouse models and human DM1 brain suggest an RNA-mediated disease mechanism whereby DMPK transcripts containing expanded CUG repeats accumulate in toxic intranuclear RNA foci that sequester Muscleblind-like (MBNL) RNA binding proteins. MBNL proteins regulate alternative splicing in differentiated cells including muscle and neurons. While RNA mis-splicing appears to be a critical driver to explain muscle phenotypes in DM1, the mechanisms that underlie neurological impairments in DM1 are not known. A few studies have identified a role for MBNLs in mRNA localization although the mechanism is not well understood. Here we report characterization of the cellular mechanism for MBNL mediated mRNA localization in differentiated cortical neurons in culture. MBNL proteins were localized in a punctate distribution in axons and dendrites. Live cell imaging of GFP tagged cytoplasmic MBNL isoforms revealed transport granules that exhibited bidirectional transport in axons and dendrites. We used the split kinesin assay in Neuro2A cells to screen thirteen kinesin tail domains. MBNL proteins were strongly recruited by the tail domain of Kif1bα. Other kinesin tails exhibited no interaction or weak localization activity. Using double label live cell imaging of fluorescently tagged proteins in cortical neurons, we observed, in neurites, co-transport of MBNL with both Kif1bα and Kif1C, which contain similar carboxy terminal tail domains. CLIP-Seq analysis has documented the presence of multiple MBNL binding motifs in the 3’UTR of SNAP25 mRNA. Cortical neurons expressing DMPK transcripts containing 480 CUG repeats display mislocalization of SNAP25 mRNA, with transcripts being abnormally restricted to the somatic compartment. Nuclear retention of SNAP25 mRNA is also an apparent phenotype observed in repeat containing cortical neurons. Overexpression of HA-MBNL via AAV transduction in mature cortical neurons expressing 480 repeat-containing DMPK transcripts appears to rescue both nuclear retention and mislocalization of SNAP25 mRNA, restoring distribution of SNAP25 mRNA to neurite compartments. The kinesin tails were once more utilized, but as potential dominant negatives associating with endogenous MBNL, and, due to lack of a motor domain, preventing MBNL from localizing its mRNA targets. We found that the Kif1Bα and Kif1C tails prevented normal localization of SNAP25 mRNA to neurites, instead being largely retained in the soma of cortical neurons, whereas the other kinesin tails did not noticeably affect SNAP25 mRNA localization. An association of Kif1bα with RNA transport granules has not been previously reported. Finally, in collaboration of Eric Wang, from the University of Florida, MBNL mutants have been developed and utilized in the above experiments to characterize the domain(s) necessary for proper mRNA localization. In sum, these results suggest a new function for MBNL-Kif1bα interactions in mRNA localization that may be impaired in myotonic dystrophy, possibly contributing to CNS symptoms.

 

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Graduate Studies

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Keywords
Phd proposal
Status
  • Created By: Tatianna Richardson
  • Workflow Status: Published
  • Created On: Jan 21, 2021 - 11:06am
  • Last Updated: Jan 21, 2021 - 11:15am