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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.

Recent Lab News

Congratulations to Sager and Shawn! Their Molecular Cell paper is now online. In this study, we reveal the patterns of RNA secondary structure and RNA-binding protein interaction sites in the Arabidopsis nucleus. It is online now at: For those interested in the datasets and affiliated data browsers please go to the following link:

Very nice press release about our recent Molecular Cell paper can be found at the following site:

Collaborative study with Chen lab to find Pol IV transcripts in Arabidopsis published in Genome Research. If interested, paper can be found here: doi: 10.1101/gr.182238.114.

Methods paper describing the use of PIP-seq to profile RBP-binding sites transcriptome-wide in HEK293T cells. Those interested can find the paper here: doi: 10.1016/j.ymeth.2014.10.021. The RBP binding site lists are provided as Supplemental Tables for those that are interested in those datasets. This is Ian Silverman's second first author paper from the lab. Congrats Ian!

Yih-Chii's second first author paper published in Bioinformatics. Congrats Yih-Chii! In this manuscript, we describe HIPPIE, which is an analytical pipeline for identifying enhancer-promoter pairs using Hi-C datasets. Paper can be found here: doi: 10.1093/bioinformatics/btu801.

Collaborative study focusing on a quality control system that monitors microRNA biogenesis in mammals with the Mourelatos lab published in Mol. Cell . If interested the paper can be found here:

Congratulations to Dr. Fan Li on receiving the the Saul Winegrad Award for Outstanding Dissertation for the GCB program!

Congratulations to our award winning undergraduate Sarah Foster on he Churchill Scholarship! Another wonderful accomplishment by a fantastic student. For more information on Sarah's awards see the following news article:

Congratulations to Ian and Fan on their recent Genome Biol. paper where we describe PIP-seq, which is our novel approach for transcriptome-wide identification of protein-RNA interaction sites in eukaryotic transcriptomes. This manuscript can be found here:

Nice Penn News piece about our novel PIP-seq methodology can be found here:

Congratulations to Ian Silverman! He has won a poster prize at both the CAMB and Genetics Dept. Symposiums this fall by presenting his work on PIP-seq development, which is a global RNA-binding protein footprinting technique. His paper describing this work is coming soon!

A huge congratulations to Dr. Fan Li, who successfully defended his thesis yesterday. Welcome to the Ph.D. club, Fan!

Congratulations to Paul and Fanny on their Methods paper now online. This manuscript provides a detailed description on how to utilize our software CoRAL for classifying RNAs using RNA sequencing data.

Congratulations to Paul, Fanny, and Ian on their RNA paper now online. This study describes our development of the software that we call HAMR, which can be used to identify and annotate many different kinds of modification sites in RNA molecules using high-throughput RNA sequencing data.

Collaborative study with the Weiss lab on transcriptomic analysis of murine coronavirus-infected mice spinal cords published in PLoS ONE

Collaborative epigenomics study with the Berger lab online now at Genes Dev. See Publications for citation.

Congratulations to Matthew and Nate on their paper in Methods where we have made major improvements to our GMUCT protocol. The manuscript describes GMUCT 2.0, which we use on Arabidopsis flower buds and 3 different human cell lines.

Congrats to Fanny Leung and Paul Ryvkin on their paper online now at Nucleic Acids Research where the Gregory, Ungar, and Wang labs collaborated to develop a method that allows more comprehensive classification of non-coding RNAs using smRNA-seq data. The paper can be found here:

Congratulations to Lee on his recent Plant Signaling and Behavior paper were we show that Arabidopsis mRNA secondary structure likely also has effects on protein maturation and expression.

Congratulations to undergraduate research assistant Sarah Foster on being named a Goldwater Scholar! We are all so very proud of your wonderful accomplishment.

Congratulations to Anissa Alexander on her NSF predoctoral fellowship award, and to Lee Vandivier for being named an honorable mention for this prize. We are all very proud of both of you!

We would like to thank the NSF (and our PO Dr. Susannah Gal) for the new grant award that will allow us to analyze RNA-protein interaction sites transcriptome-wide in multiple plant species.

Congrats to Yih-Chii Hwang and Qi Zheng on their paper online now at Nucleic Acids Research where the Gregory and Wang labs collaborated to develop a method that allows comprehensive identification of enhancer - promoter interactions using Hi-C data. The paper can be found here: ijkey=j5Pz4MCJjwUL3ho&keytype=ref

Nice article about the research in the Gregory lab can be found here:

Collaborative study on vsiRNA processing by Drosophila melanogaster Dcr-2 with the Cherry lab now published in PLoS ONE. The paper can be found here:

Congrats to Ian and Fan for their nice review about plant RNA secondary structure and RNA-binding proteins for Plant Science that is now available online at:

Congratulations to Fan and Qi on their recently accepted Plant Cell paper. In this study, we uncovered that RNA secondary structure has significant regulatory effects on gene expression in Arabidopsis thaliana. If interested, you can find the paper here:

Collaborative study with the Wierzbicki lab on AGO4 targeting in Arabidopsis now online at The Plant Journal.

Collaborative study with the Mourelatos lab on piRNA biogenesis and function now online at Nature Structural and Molecular Biology.

Congratulations to Lee on his Cell and Molecular Biology T32 Award. Congrats, Lee!

We thank the Gordon and Betty Moore Foundation for their support to the Gregory and Lyon labs to fund the development of the EPIC-CoGe Browser, which will be an easy-to-use web-based system to store, access, and visualize Arabidopsis epigenetic data in a comparative genomics context. For more information, see this press release (

For anybody interested in the structure score data for Drosophila and C. elegans mRNAs, all of this data is available for download here:

Congratulations to Ian on his Genetics T32 Award. Congrats, Ian!

Fan and Paul's paper on a new web-based tool for RNA-seq analysis in the context of secondary structure (SAVoR) is now online at Nucleic Acids Research: doi: 10.1093/nar/gks310.

Congratulations to Fan and Qi for their paper in Cell Reports on global analysis of secondary structure in Drosophila and C. elegans!!!

If you are interested in accessing our high-throughput RNA sequencing data and models of RNA secondary structure, please visit our webbrowsers at:

Welcome our new lab members, Ian, Nate and Ammar!

Gregory Lab awarded NSF Career Award for characterizing RDR substrates.

Brian D. Gregory
Assistant Professor
Department of Biology
University of Pennsylvania

433 S. University Ave
Lynch Laboratories
Room 204G (office)
Room 104 (wet lab)
Room 218 (dry lab)
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