Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Rout Laboratory

Abstract

During and after transcription in the nucleus, messenger RNAs (mRNAs) undergo a variety of processing events before being exported to the cytoplasm through the nuclear pore complex. mRNA processing and nuclear export require a wide range of protein factors, which interact with maturing transcripts and each other to form dynamic mRNP complexes. While there are many core, essential mRNP factors, the pathways governing mRNA maturation are not uniform, and different transcripts can be associated with mRNP complexes of dramatically different composition or kinetics. To date though, it has been difficult to study RNP complexes specific to any single mRNA species, as each transcript is relatively unabundant in the cell, and few robust techniques exist to specifically purify a particular mRNP for proteomic analysis. We thus sought to develop a method to isolate mRNPs from a single transcript, allowing us to study the dynamic RNP compositions of individual mRNA maturation pathways. To optimize purifications of the protein tags required for RNP isolations, we first generated high affinity reagents targeting key tags like GFP and mCherry. Instead of traditional antibodies, we chose to use nanobodies: recombinant single domain derivatives of a heavy chain-only antibody variant found in camelids. The recombinant nature and small size of nanobodies make them ideal reagents for affinity isolations. We developed an improved pipeline for the identification of nanobody repertoires against any antigen of interest, which provided us with 25 nanobodies against GFP, the most common and robust protein tag in use. This pipeline has also allowed us to develop nanobodies against a variety of other antigens of biomedical interest. With the help of optimized reagents, we developed a two-step purification method allowing highly targeted isolations of mRNPs, starting in a budding yeast model system. In our approach, a single target transcript is tagged with MS2 hairpin sequences – these hairpins are bound specifically and with high affinity by the bacteriophage MS2 coat protein (MS2CP). In the first purification step, a chosen RNP protein known to be associated with a particular mRNA processing step of interest is Protein A-tagged and affinity isolated. From this material, anti-GFP nanobodies are used in the second step to isolate the MS2-tagged transcript of interest, through purification of MS2CP-GFP fusion proteins bound to the tag. This approach is able to efficiently and cleanly isolate a particular transcript at a chosen step of mRNP maturation. The use of an RNP factor as a separate purification target both improves overall purity and simplifies analysis by limiting heterogeneity of the mRNP mixture. Using this novel method for single mRNP isolations, we have performed a preliminary survey of transcripts with distinct sequence elements suspected to be associated with unique processing machinery. Mass spectrometric (MS) analysis of RNPs co-purified with these transcripts revealed several RNA-specific changes in composition. Most notably, introns from either a house keeping ACT1 gene or the RPS30b ribosomal protein gene led to dramatically different levels of various splicing-related proteins. These differences provide mechanistic insight into changes in the kinetics of spliceosome assembly determined by intron sequence.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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