Deciphering the role of N6-methyladenosine (m6A) on mRNA during adaptive stress

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

ABSTRACT Prostate cancer (PrCa) is the second leading cause of cancer related deaths in men. Current treatment methods are no longer effective once cancer cells become resistant, allowing cells to progress to metastasis. Two markers for worse patient outcomes in PrCa are the Integrated Stress Response (ISR) and the RNA modification N6- methyladenosine (m6A). The ISR is an adaptive pathway that can be triggered by many stressors within cancer cells and the tumor microenvironment. This pathway can be activated by cancer treatments and its activation can lead to drug resistance and metastasis. The ISR serves as a protective mechanism that limits extraneous protein synthesis and promotes the translation of select stress response and pro-survival transcripts. The mechanism by which this adaptive translation occurs is relatively unknown, but one method by which mRNAs may be selected for increased translation is through m6A. m6A methylation has been observed to alter the splicing, degradation, localization, and translation of mRNAs. In PrCa cell lines, inhibiting m6A deposition leads to growth deficiencies. Recent papers have tied the m6A methylation of specific transcripts to their translation status during the ISR. The goal of this proposal is to determine if m6A modifications on select transcripts are required for their translation during the ISR, and whether blocking m6A will sensitize PrCa cells to targeted therapies. Here, two aims are proposed to determine the role of m6A modifications in marking transcripts for translation during the ISR and to identify how the m6A machinery can drive PrCa. In Aim 1, m6A sequencing will be carried out from polysome fractions with and without inhibition of the ISR to correlate the m6A methylation state of a transcript during the ISR to its translation status. The top targets identified by the m6A sequencing will be validated using a knockout of m6A writer METTL3 and qPCR of mRNAs derived from polysome profiles. Potential impacts on mRNA degradation rates will also be determined by inhibiting transcription and analyzing transcripts with and without the presence of m6A. Additionally, isolation of proteins bound to specific regions of top target mRNAs followed by mass spectrometry with and without m6A will be employed to determine which proteins bind m6A transcripts during the ISR. In Aim 2, four m6A regulatory proteins—VIRMA, eIF3h, YTHDF3, and hnRNPA2B1—found to be significantly upregulated in PrCa datasets and disease progression, will be evaluated. Correlations between their expression profile and oncogenic potential in metastatic PrCa cells, alongside treatment with current PrCa therapies, will be analyzed in cells with stable knockdown and overexpression of these genes. Lastly, the mechanism by which the proteins function will be established by identifying the mRNAs they bind to and the possible downstream consequences of their binding on mRNA translation during the ISR. Pursuing these aims will enable a detailed representation of the role m6A plays in driving select mRNA translation during the ISR. Gaining an increased understanding of the drivers of selective translation may provide novel insights into targeting strategies for treating advanced PrCa.