16 research outputs found
HiTRACE-Web: an online tool for robust analysis of high-throughput capillary electrophoresis
To facilitate the analysis of large-scale high-throughput capillary
electrophoresis data, we previously proposed a suite of efficient analysis
software named HiTRACE (High Throughput Robust Analysis of Capillary
Electrophoresis). HiTRACE has been used extensively for quantitating data from
RNA and DNA structure mapping experiments, including mutate-and-map contact
inference, chromatin footprinting, the EteRNA RNA design project and other
high-throughput applications. However, HiTRACE is based on a suite of
command-line MATLAB scripts that requires nontrivial efforts to learn, use, and
extend. Here we present HiTRACE-Web, an online version of HiTRACE that includes
standard features previously available in the command-line version as well as
additional features such as automated band annotation and flexible adjustment
of annotations, all via a user-friendly environment. By making use of
parallelization, the on-line workflow is also faster than software
implementations available to most users on their local computers. Free access:
http://hitrace.or
Exploring the global adoption of citizen science
In recent years there has been a growing interest toward the application of Web-based citizen science platforms. Such platforms use crowdsourcing techniques to support scientific advancements, and in several cases, have lead to new scientific discoveries which were not originally considered. Our work explores the highly successful Web-based citizen science platform, Zooniverse, a crowdsourcing platform with a userbase of over 1 million participants who volunteer their free time to support scientific enquiries. We focus on the growth of the Zooniverse platform, which has evolved from a rudimentary crowdsourcing platform where users were presented with tasks, into a platform which has become a rich community of citizen scientists, discussion, and interaction. Building upon existing research into the motivations and design considerations of developing and sustaining citizen science projects, this paper explores the space of citizen science engagement within the Zooniverse, and ask the question of whether citizen science has become a worldwide activity
A mutate-and-map protocol for inferring base pairs in structured RNA
Chemical mapping is a widespread technique for structural analysis of nucleic
acids in which a molecule's reactivity to different probes is quantified at
single-nucleotide resolution and used to constrain structural modeling. This
experimental framework has been extensively revisited in the past decade with
new strategies for high-throughput read-outs, chemical modification, and rapid
data analysis. Recently, we have coupled the technique to high-throughput
mutagenesis. Point mutations of a base-paired nucleotide can lead to exposure
of not only that nucleotide but also its interaction partner. Carrying out the
mutation and mapping for the entire system gives an experimental approximation
of the molecules contact map. Here, we give our in-house protocol for this
mutate-and-map strategy, based on 96-well capillary electrophoresis, and we
provide practical tips on interpreting the data to infer nucleic acid
structure.Comment: 22 pages, 5 figure
Nat Biotechnol
We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.DP1 HG007811/HG/NHGRI NIH HHS/United StatesT32 HG000035/HG/NHGRI NIH HHS/United States1DP1HG007811/DP/NCCDPHP CDC HHS/United States5T32HG000035/HG/NHGRI NIH HHS/United States2016-03-01T00:00:00Z26237516PMC4564351vault:1379
COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures
SummaryThere is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription
Short- and long-range interactions in the HIV-1 5′ UTR regulate genome dimerization and packaging
RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5′ untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5′ UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS–SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.Peer Reviewe