Thread by @thatdnaguy

#ASHG20 OSR
Coding regions affect mRNA stability in human cells.
Olivia S. Rissland.

#ASHG20 OSR Want to understand gene expression in eukaryotic cells. Especially coupling of translation and mRNA decay.

#ASHG20 OSR Two main avenues. 1) mechanistic studies. 2) regulation of mRNA decay and translation during development.

#ASHG20 OSR Different parts of an mRNA have parts that play different roles in regulation. UTRs can affect gene expression. Upstream ORFs (uORF). secondary structure, microRNA binding sites, etc. open-reading frame mostly considered passive part of process.

#ASHG20 OSR Think that the main ORF actually helps control RNA stability. Ribosome acts as a sensor for mRNA decay. Helps recognize aberrant transcripts. But even normal translation helps ribosome act as sensor via elongation speeds. Affected by codon optimization.

#ASHG20 OSR Low optimality leads to slow elongation and can help trigger transcript degeneration.

#ASHG20 OSR Codon optimality affect by tRNA abundance, codon usage in transcriptome, and wobble position interactions. Given these multiple factors can actually vary by tissue and developmental stage.

#ASHG20 OSR What is the role of the open reading frame in mRNA decay within *human* cells?

#ASHG20 OSR One issue is that ORFs and UTRs co-evolve over time. Makes assigning causality to UTR over ORF or vice versa can be difficult.

#ASHG20 OSR The human ORFeome collection can be used to determine the role of the ORF in mRNA stability ( b/c collection has the ORFs in invariant UTRs). Made pools. Lentiviral transduction. Stable lines. Then mRNA half-life determination with metabolic labeling.

#ASHG20 OSR Only works for ORFs that aren't expressed endogenously. Does changing ORF affect stability (as measured by mRNA half-life)? ORFeome mRNAs tend to be more stable than endogenous RNA.

#ASHG20 OSR Large variability of mRNA halflife for ORFeome mRNAs. Has as much variation in stability as endogenous mRNAs. Suggests just changing ORF gives full range of mRNA half-lives.

#ASHG20 OSR Is the affect via translation, i.e. is translation required for the difference? If you treat with DMSO and 4EGI-1. With 4EGI-1 see lower half-life and reduced variation. At least some of the effect is mediated by translation.

#ASHG20 OSR What ORF features mediate stability? Codon optimality might be a critical contributor. Checked the codon stability coefficient. Find codon use is associated with ORFeome mRNA stability.

#ASHG20 OSR [ instant extension to human genetics - even synonymous variants could affect codon optimality, altering transcript stability and perhaps altering function ]

#ASHG20 OSR Significant correlation with elongation speed and the codon stability coefficient. Is it a general trend? Reproducible in several datasets and different cell lines. Doesn't work the same in mouse cell line [ which isn't expected to have some codon optimality ].