Non-canonical Messenger RNA Translation in Human Cells.
Faculty Mentor(s) Name(s)
Dr. Arnab Sengupta
Abstract
In eukaryotes, including humans, translation of the genetic message is an essential process that needs to be carefully controlled. Cells have procedures to measure conditions and can accordingly promote or block mRNA translation for protein synthesis. Normally, mRNA translation requires a series of steps - (a) recognizing the mRNA 5’ end, (b) assembly of translation factors and the pre-initiation complex, and (c) scanning to find the start signal. This multi-step initiation process allows efficient control of translation, a resource-hungry process that is turned off when cells have other priorities. However, some genes defy the cells' priorities and continue making proteins when most genes have been turned off due to cellular stress. This phenomenon is broadly defined as non-canonical translation. Our goal is to investigate the role of RNA structure and interactions in non-canonical translation. To achieve this, we apply the SHAPE-MaP chemical probing strategy – a technology that allows us to determine the folding patterns of regulatory RNA regions. Here we describe the SHAPE-MaP methodology and present structure probing data for a control RNA and a target regulatory RNA, the RPA2 (Replication protein A2) mRNA 5’untranslated region. RPA2 has been reported to undergo non-canonical translation however the mechanism remains poorly described. Our work aims to fill a critical gap in understanding the regulation of RPA2 gene expression, which not only plays a role in normal cellular processes but is also implicated in cancer development. Our work is still at a preliminary stage, but already we have implemented many innovative technologies including human cell culture, RNA extraction & quantitation, reverse-transcription, PCR, library preparation, and next-generation sequencing. In future experiments, we will compare initial RNA structure analysis with live cell conditions under distinct types of cellular stress. We also describe the experimental design for an upcoming validation study using dual luciferase reporter assays.
Start Date
27-3-2024 9:00 AM
End Date
27-3-2024 9:50 AM
Location
Magnolia Ballroom
Non-canonical Messenger RNA Translation in Human Cells.
Magnolia Ballroom
In eukaryotes, including humans, translation of the genetic message is an essential process that needs to be carefully controlled. Cells have procedures to measure conditions and can accordingly promote or block mRNA translation for protein synthesis. Normally, mRNA translation requires a series of steps - (a) recognizing the mRNA 5’ end, (b) assembly of translation factors and the pre-initiation complex, and (c) scanning to find the start signal. This multi-step initiation process allows efficient control of translation, a resource-hungry process that is turned off when cells have other priorities. However, some genes defy the cells' priorities and continue making proteins when most genes have been turned off due to cellular stress. This phenomenon is broadly defined as non-canonical translation. Our goal is to investigate the role of RNA structure and interactions in non-canonical translation. To achieve this, we apply the SHAPE-MaP chemical probing strategy – a technology that allows us to determine the folding patterns of regulatory RNA regions. Here we describe the SHAPE-MaP methodology and present structure probing data for a control RNA and a target regulatory RNA, the RPA2 (Replication protein A2) mRNA 5’untranslated region. RPA2 has been reported to undergo non-canonical translation however the mechanism remains poorly described. Our work aims to fill a critical gap in understanding the regulation of RPA2 gene expression, which not only plays a role in normal cellular processes but is also implicated in cancer development. Our work is still at a preliminary stage, but already we have implemented many innovative technologies including human cell culture, RNA extraction & quantitation, reverse-transcription, PCR, library preparation, and next-generation sequencing. In future experiments, we will compare initial RNA structure analysis with live cell conditions under distinct types of cellular stress. We also describe the experimental design for an upcoming validation study using dual luciferase reporter assays.