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dc.contributor.advisorMelissa J. Moore, PhD
dc.contributor.authorHeyer, Erin E.
dc.date2022-08-11T08:08:45.000
dc.date.accessioned2022-08-23T16:07:06Z
dc.date.available2022-08-23T16:07:06Z
dc.date.issued2015-10-06
dc.date.submitted2016-04-04
dc.identifier.doi10.13028/M2GK57
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32180
dc.description.abstractDeep 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.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectDissertations, UMMS
dc.subjectHigh-Throughput Nucleotide Sequencing
dc.subjectNucleotides
dc.subjectRNA
dc.subjectGene Library
dc.subjectOpen Reading Frames
dc.subjectHigh-Throughput Nucleotide Sequencing
dc.subjectNucleotides
dc.subjectRNA
dc.subjectGene Library
dc.subjectOpen Reading Frames
dc.subjectBiochemistry
dc.subjectBioinformatics
dc.subjectCell Biology
dc.subjectComputational Biology
dc.subjectGenetics
dc.subjectMolecular Biology
dc.subjectMolecular Genetics
dc.titleOptimizing RNA Library Preparation to Redefine the Translational Status of 80S Monosomes: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1812&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/810
dc.legacy.embargo2017-01-06T00:00:00-08:00
dc.identifier.contextkey8420505
refterms.dateFOA2022-08-25T04:01:26Z
html.description.abstract<p>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.</p>
dc.identifier.submissionpathgsbs_diss/810
dc.contributor.departmentRNA Therapeutics Institute
dc.description.thesisprogramBiochemistry and Molecular Pharmacology


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