Genomics and regulatory functions of microRNAs and small silencing RNAs in Arabidopsis thaliana

Small RNA-mediated gene silencing is a mechanism widely employed by eukaryotes to repress many loci including some involved in critical developmental transitions. In plants, endogenous small RNAs consist of two broad classes, the ~21-nucleotide (nt) microRNAs (miRNAs) and the diverse ~22-24-nt trans-acting and heterochromatic short interfering RNAs (siRNAs). In order to more extensively characterize the small RNA landscape in plants and to identify undiscovered miRNAs and siRNAs, we performed high-throughput sequencing of small RNAs. We generated a large dataset consisting of >340,000 unique sequences expressed in several representative plant organs and developmental stages including wild-type seedlings, flowers, leaves and siliques. Application of enhanced miRNA annotation criteria gleaned from the data revealed the existence of at least 38 apparently recently-evolved miRNAs that were much less abundant in plant tissues than the 26 conserved miRNA families, and had a greater diversity of predicted target genes. We characterized several of these miRNAs more closely. Our results supported a homeostatic auto-regulatory loop for DCL1 via the intron-embedded miR838, and elaborated on the prevailing model of DCLl-mediated miRNA biogenesis with the finding that at least two miRNAs (miR839 and miR822) are processed exclusively by DCL4. Several microRNA target sites were experimentally validated, including the miR823-directed cleavage of the DNA cytosine methylation factor CHROMOMETHYLASE3. We also identified a trans-acting siRNA-generating locus which we called TAS4, and confirmed that miR828 triggers phased siRNA production by specifying targeted cleavage of TAS4 transcripts.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Rajagopalan, Ramya

Advisor dc:contributor.advisor

• David P. Bartel.

• 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/1721.1/43787

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/43787