Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation

  1. Christian K Roy
  2. Sara Olson
  3. Brenton R Graveley
  4. Phillip D Zamore
  5. Melissa J Moore  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Massachusetts Medical School, United States
  2. University of Connecticut Health Center, United States

Abstract

Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increases the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. Here, we describe SeqZip, a methodology that uses RNA-templated DNA-DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1 and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.

Article and author information

Author details

  1. Christian K Roy

    RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.
  2. Sara Olson

    Institute for Systems Genomics, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, United States
    Competing interests
    No competing interests declared.
  3. Brenton R Graveley

    Institute for Systems Genomics, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, United States
    Competing interests
    No competing interests declared.
  4. Phillip D Zamore

    RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    Phillip D Zamore, Reviewing Editor, eLife.
  5. Melissa J Moore

    RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    melissa.moore@umassmed.edu
    Competing interests
    No competing interests declared.

Reviewing Editor

  1. Aviv Regev, Broad Institute of MIT and Harvard, United States

Version history

  1. Received: June 15, 2014
  2. Accepted: April 12, 2015
  3. Accepted Manuscript published: April 13, 2015 (version 1)
  4. Version of Record published: May 26, 2015 (version 2)

Copyright

© 2015, Roy et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,347
    views
  • 761
    downloads
  • 27
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Christian K Roy
  2. Sara Olson
  3. Brenton R Graveley
  4. Phillip D Zamore
  5. Melissa J Moore
(2015)
Assessing long-distance RNA sequence connectivity via RNA-templated DNA-DNA ligation
eLife 4:e03700.
https://doi.org/10.7554/eLife.03700

Share this article

https://doi.org/10.7554/eLife.03700

Further reading

    1. Chromosomes and Gene Expression
    2. Immunology and Inflammation
    Rajan M Thomas, Matthew C Pahl ... Andrew D Wells
    Research Article

    Ikaros is a transcriptional factor required for conventional T cell development, differentiation, and anergy. While the related factors Helios and Eos have defined roles in regulatory T cells (Treg), a role for Ikaros has not been established. To determine the function of Ikaros in the Treg lineage, we generated mice with Treg-specific deletion of the Ikaros gene (Ikzf1). We find that Ikaros cooperates with Foxp3 to establish a major portion of the Treg epigenome and transcriptome. Ikaros-deficient Treg exhibit Th1-like gene expression with abnormal production of IL-2, IFNg, TNFa, and factors involved in Wnt and Notch signaling. While Ikzf1-Treg-cko mice do not develop spontaneous autoimmunity, Ikaros-deficient Treg are unable to control conventional T cell-mediated immune pathology in response to TCR and inflammatory stimuli in models of IBD and organ transplantation. These studies establish Ikaros as a core factor required in Treg for tolerance and the control of inflammatory immune responses.

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Lucie Crhak Khaitova, Pavlina Mikulkova ... Karel Riha
    Research Article

    Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.