RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP)

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Many biological processes are RNA-mediated, but higher-order structures for most RNAs are unknown, making it difficult to understand how RNA structure governs function. Here we describe SHAPE mutational profiling (SHAPE-MaP) that makes possible de novo and large-scale identification of RNA functional motifs. Sites of 2’-hydroxyl acylation by SHAPE are encoded as non-complementary nucleotides during cDNA synthesis, as measured by massively parallel sequencing. SHAPE-MaP-guided modeling identified greater than 90% of accepted base pairs in complex RNAs of known structure and was used to define a second-generation model for the HIV-1 RNA genome. The HIV-1 model contains all known structured motifs and previously unknown elements, including experimentally validated pseudoknots. SHAPE-MaP yields accurate and high-resolution secondary structure models, enables analysis of low abundance RNAs, disentangles sequence polymorphisms in single experiments, and will ultimately democratize RNA structure analysis.

Related collections

Most cited references 48

Record: found
Abstract: found
Article: not found

FLASH: fast length adjustment of short reads to improve genome assemblies.

T. Magoc, S. L. Salzberg (2013)

Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.

0 comments Cited 6058 times – based on 0 reviews      Review now

Record: found
Abstract: found
Article: not found

Architecture and Secondary Structure of an Entire HIV-1 RNA Genome

Joseph M Watts, Kristen Dang, Robert J. Gorelick … (2009)

Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS, and other serious health threats. Viral replication is regulated at many levels, including using conserved genomic RNA structures. Most potential regulatory elements within viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions. Highly used splice acceptors lie in unstructured motifs and hypervariable regions are sequestered from flanking genome regions by stable insulator helices. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by numerous previously unrecognized regulatory motifs and that extensive RNA structure may constitute an additional level of the genetic code.

0 comments Cited 354 times – based on 0 reviews      Review now

Record: found
Abstract: found
Article: not found

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.

Kevin Wilkinson, Edward Merino, Kevin Weeks (2006)

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) interrogates local backbone flexibility in RNA at single-nucleotide resolution under diverse solution environments. Flexible RNA nucleotides preferentially sample local conformations that enhance the nucleophilic reactivity of 2'-hydroxyl groups toward electrophiles, such as N-methylisatoic anhydride (NMIA). Modified sites are detected as stops in an optimized primer extension reaction, followed by electrophoretic fragment separation. SHAPE chemistry scores local nucleotide flexibility at all four ribonucleotides in a single experiment and discriminates between base-paired versus unconstrained or flexible residues with a dynamic range of 20-fold or greater. Quantitative SHAPE reactivity information can be used to establish the secondary structure of an RNA, to improve the accuracy of structure prediction algorithms, to monitor structural differences between related RNAs or a single RNA in different states, and to detect ligand binding sites. SHAPE chemistry rarely needs significant optimization and requires two days to complete for an RNA of 100-200 nucleotides.

0 comments Cited 298 times – based on 0 reviews      Review now

All references

Author and article information

Journal

Journal ID (nlm-journal-id): 101215604

Journal ID (pubmed-jr-id): 32338

Journal ID (nlm-ta): Nat Methods

Journal ID (iso-abbrev): Nat. Methods

Title: Nature methods

ISSN (Print): 1548-7091

ISSN (Electronic): 1548-7105

Publication date Nihms-submitted: 19 June 2014

Publication date (Electronic): 13 July 2014

Publication date (Print): September 2014

Publication date PMC-release: 01 March 2015

Volume: 11

Issue: 9

Pages: 959-965

Affiliations

[1 ]Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290

[2 ]Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599

Author notes

[4 ] Correspondence, weeks@ 123456unc.edu

[3]

These authors contributed equally

Article

Manuscript ID: NIHMS606325

DOI: 10.1038/nmeth.3029

PMC ID: 4259394

PubMed ID: 25028896

SO-VID: 24056b33-49b8-47ee-9ff7-a37fe05e7656

History

Comments

Comment on this article

Cited by 276

See all cited by

Most referenced authors 693

See all reference authors