Using high-throughput sequencing to study RNA secondary structure globally
We have recently developed high-throughput, sequencing-based approaches to study RNA secondary structure on a global scale. To do this, we have married classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate the pairing status of all nucleotides in the RNA molecules of eukaryotic organisms. Using this information, we are producing genome-wide collections of RNA secondary structure models for organisms of interest. We believe that the findings from these approaches highlight the importance of base-paired RNAs in eukaryotes and present an approach that should be widely applicable for the analysis of this key structural feature of RNA in any and all organisms.
We are also using these approaches to identify all small (sm)RNA-producing substrates of RNA-DEPENDENT RNA POLYMERASEs (RDRs). More specifically, we use the combination of transcriptome-wide double-stranded (dsRNA) and small RNA sequencing to interrogate the substrates of this class of enzymes in eukaryotes. We are currently characterizing the RDRs of Arabidopsis and C. elegans.
Mechanisms and regulation of RNA silencing pathways
Our research also focuses on unraveling the molecular mechanisms governing the regulation and function of RNA silencing pathways. RNA silencing is a highly conserved pathway that controls gene expression post-transcriptionally. This pathway is trigged by either the production of double-stranded RNA (dsRNA) or self-complementary fold-back structures that give rise to small RNAs (smRNAs) through the activity of DICER or DICER-LIKE (DCL) RNase III-type ribonucleases. These smRNAs comprise the sequence-specific effectors of RNA silencing pathways that direct the negative regulation or control of genes, repetitive sequences, viruses, and mobile elements. Therefore, smRNAs control diverse functions from development to immunity and their dysregulation leads to a wide-variety of diseases. smRNAs are comprised of microRNAs (miRNAs) and several classes of small interfering RNAs (siRNAs), which are differentiated from one another by their distinct biogenesis pathways, the classes of genomic loci from which they arise, and their targets. While a number of the key components of these pathways have been identified, there are many still to be isolated and characterized. Additionally, the underlying regulatory mechanisms controlling the production and targeting of specific smRNA populations are not well understood. Therefore, our research has concentrated on identifying novel proteins (including those involved in RNA stability/degradation) that are required for the metabolism of various classes of smRNAs and how these factors regulate specific RNA silencing pathways. We have demonstrated that ABH1/CBP80, a subunit of the mRNA cap-binding complex, is necessary to obtain proper mature miRNA levels, which suggests this protein is an essential component of the miRNA-mediated RNA silencing pathway. Using high-throughput sequencing technologies, we have shown that XRN4/EIN5, a 5'-3' exoribonuclease, affects the levels of a smRNA class that is processed from both sense and anti-sense strands of ~130 endogenous transcripts that are converted to double-stranded RNA and subsequently processed. Using a combination of genetics, biochemistry, and sequencing techniques, our results revealed unexpected connections between RNA metabolism and silencing pathways.
Making use of genomic, bioinformatic, and systems biology approaches with molecular genetic and biochemical techniques we are identifying and characterizing additional components required for the metabolism of various classes of smRNAs, as well as proteins involved in the regulation of specific smRNA populations and RNA silencing pathways. Specifically, our lab is taking a forward genetic approach using the model genetic organism Arabidopsis thaliana to identify new factors, and have already identified candidate genes that we are characterizing using genomic, molecular biological, and cell biological techniques in Arabidopsis. Furthermore, as these pathways are highly conserved, we are also studying these factors in smRNA pathways in animal models. The findings from this work will allow a better understanding of how RNA silencing pathways function, and the ways they can be manipulated for controlling gene expression across eukaryotic systems.
Brian D. Gregory
Department of Biology
University of Pennsylvania
433 S. University Ave
Room 204G (office)
Room 104 (wet lab)
Room 218 (dry lab)
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