Poster abstracts
Poster number 65 submitted by Akash Macha
Exploring the functions of conserved RNA structures in the human Titin 5’ UTR
Akash Macha (Department of Biological Sciences, Carnegie Mellon University), Gemma May (Department of Biological Sciences, Carnegie Mellon University), Joel McManus (Department of Biological Sciences, Carnegie Mellon University)
Abstract:
The human TITIN gene (TTN) encodes an enormous protein whose translation
poses extraordinary challenges for the ribosome. Translation is primarily controlled at
the initiation stage, in which ribosomes are recruited to mRNA start codons to begin
protein synthesis. A variety of cis-acting mRNA sequences and structures in 5’ UTRs
affect ribosome recruitment. For example, upstream Open Reading Frames (uORFs)
typically reduce ribosome loading on the main ORF start codon. RNA structures can
have both positive and negative influences on initiation depending on their location
relative to other 5’ UTR elements. Such structures can reduce pre-initiation complex
(PIC) loading by occluding the 5’ m7G cap, impede PIC directional scanning, and alter
the frequency of initiation at uORF and main ORF start codons. However, the relative
effects of structures and uORFs, and their potential interactions remain poorly
characterized for the vast majority of mammalian 5’ UTRs. TTN has one of the most
conserved 5’ UTRs in mammals, including a dual start codon uORF and three stemloop
structures (SL1, SL2 and SL3). Previous work in the lab suggests these stemloops
reduce translation of TTN. We used dual luciferase reporter assays to further test the
roles of the three stemloops in translation. RNA-fold was used to design mutations
expected to disrupt SL1 and compensatory mutations to restore its structure. We also
tested the positional significance of SL2 and SL3 by swapping the positions of the stem
loops. Finally, we inserted a 10xCAA sequence between SL2 and the uORF to test
whether it affects uORF binding. Our results revealed that swapping SL2 and SL3
reduced translation of luciferase and was, thus, a more inhibitory configuration.
However, SL3 is located directly before the start codon and the swap likely changed the
Kozak context to an unfavorable sequence, causing the observed decrease in luciferase
translation.
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