Biochemistry and Molecular Pharmacology
RNA Therapeutics Institute
First Thesis Advisor
Melissa J. Moore, PhD
High-Throughput Nucleotide Sequencing, Nucleotides, RNA, Gene Library, Open Reading Frames
Dissertations, UMMS; High-Throughput Nucleotide Sequencing; Nucleotides; RNA; Gene Library; Open Reading Frames
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. This optimized method was used in an adapted ribosome-profiling approach to sequence mRNA footprints protected either by 80S monosomes or polysomes in S. cerevisiae. Contrary to popular belief, we show that 80S monosomes are translationally active as demonstrated by strong three-nucleotide phasing of monosome footprints across open reading frames. Most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on upstream ORFs, canonical ORFs shorter than ~590 nucleotides and any ORF for which the total time required to complete elongation is substantially shorter than the time required for initiation. Additionally, endogenous NMD targets tend to be monosome-enriched. Thus, rather than being inactive, 80S monosomes are significant contributors to overall cellular translation.
Heyer, EE. Optimizing RNA Library Preparation to Redefine the Translational Status of 80S Monosomes: A Dissertation. (2015). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 810. DOI: 10.13028/M2GK57. http://escholarship.umassmed.edu/gsbs_diss/810
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