The Role of RNA Post-Transcriptional Modifications in RNA Sorting

  • Deema Islayem

Student thesis: Master's Thesis

Abstract

Epitranscriptome is an emerging field that study and manipulate the biochemical modification that alters the functional behavior of RNAs. Though numerous advances have been made in recent years, our understanding of the epitranscriptome is still in its infancy. RNA posttranscriptional modification adds another layer of information that rely on regulating gene expression rather than changing genetic code and sequence. m5C, one of the main RNA methylation types is regulated through NSun2 that methylate cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. The molecular role of NSun2 varies based on the class of RNA that it modifies. Based on previous work, it is highly expected that m5C play a role in RNA sorting into exosomes, the process in which cells follow to secrete selected RNA molecules into exosomes for particular biological functions. Among these functions are tissue regeneration and tumorigenesis. Different classes of RNA could be found in exosomes and they range from messenger RNAs to non-coding RNAs. However, until now the exact mechanism that regulate RNA sorting is not fully understood. In this thesis, it is studied whether RNA post-transcriptional modification specifically m5C, play a critical role in guiding specific classes of RNA to be selectively sorted into exosomes or not. NSUN2, the main methyltransferase writer of m5C is knocked down in Human Dermal Fibroblasts, followed by exosomal isolation from cell culture media to extract its RNA. Exosomal RNA is bisulfite treated and sequenced to map for m5C along its transcriptome and study the methylation status in both control and experimental exosomal groups. Preliminary results confirmed the isolation method for exosomes based on molecular, biophysical, and genetic characterization. In addition, the efficiency of NSun2 Knockdown is evaluated and proved to be working successfully in removing m5C and decreasing the expression of NSun2 in RNA and protein levels. It is the first time that m5C is studied and applied on exosomal RNA, and the results expected to be promising as it will open the door for a new discovery on the role of m5C mediated NSun2.
Date of AwardDec 2021
Original languageAmerican English

Keywords

  • RNA posttranscriptional modification
  • m5C
  • exosomes
  • RNA sorting
  • methylation
  • NSun2 Knockdown.

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