Dicer deletion and short RNA expression analysis in mouse embryonic stem cells

RNA interference (RNAi) manages many aspects of eukaryotic gene expression through sequence-specific interactions with RNA. Short RNAs, 20-30 nucleotides in length, guide the various effector proteins of RNAi to silence fully or partially complementary targets. The sequencing of endogenously expressed short RNA species coupled with genetic studies in various experimental organisms has revealed a role for RNAi in the silencing of protein-coding genes and repetitive elements in genomes. In mammals, it is unknown to what extent RNAi is involved in silencing processes other than the modulation of protein-coding gene expression, which is achieved through a class of short RNAs termed microRNAs (miRNAs). The work in this thesis quantitatively describes the short RNAs expressed in mouse embryonic stem (ES) cells. ES cell lines are derived from the pre-implantation blastocyst and can be cultured in vitro for extended periods while still maintaining pluripotency. It was demonstrated that approximately 130,000 5' phosphorylated short RNA molecules are present in a single ES cell. 10% of these short RNAs represent non-random fragments of larger, abundant non-coding RNA species, and have no known function. Low abundance short RNAs were discovered that cluster bidirectionally around the transcription start sites of protein-coding genes. These RNAs associate with features of active transcription, and may be evidence of widespread bidirectional initiation and pausing of RNA polymerase II in ES cells. There are on the order of 300 different miRNA species expressed in ES cells, comprising 85% of the total pool of 130,000 5' phosphorylated short RNAs. Based on experiments correlating miRNA abundance to target repression, only about 30 of these miRNAs are expected to carry significant ES cell regulatory capacity.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Calabrese, Joseph Mauro

Advisor dc:contributor.advisor

• Philip A. Sharp.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/42401

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/42401