Science Forum: Considerations when investigating lncRNA function in vivo

  1. Andrew R Bassett  Is a corresponding author
  2. Asifa Akhtar
  3. Denise P Barlow
  4. Adrian P Bird
  5. Neil Brockdorff
  6. Denis Duboule
  7. Anne Ephrussi
  8. Anne C Ferguson-Smith
  9. Thomas R Gingeras
  10. Wilfried Haerty
  11. Douglas R Higgs
  12. Eric A Miska
  13. Chris P Ponting  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. Max-Planck-Institut für Immunbiologie und Epigenetik, Germany
  3. Research Center for Molecular Medicine of the Austrian Academy of Sciences, Austria
  4. University of Edinburgh, United Kingdom
  5. Ecole Polytechnique Fédérale Lausanne, Switzerland
  6. European Molecular Biology Laboratory, Germany
  7. University of Cambridge, United Kingdom
  8. Cold Spring Harbor Laboratory, United States
  9. Weatherall Institute of Molecular Medicine, United Kingdom
3 figures and 1 table

Figures

lncRNAs can act through cis and/or trans mechanisms. lncRNAs (pink) can act to regulate expression of their genomically neighbouring protein-coding genes (black) in cis (upper panel), or of distant protein-coding genes in trans (lower panel).

In both situations, the RNA moiety itself may act through binding to cellular proteins (blue ovals) or via base-pairing with other RNAs (blue stem-loop) to modulate their function or binding. The RNA may also directly bind double-stranded DNA in trans (Grote et al., 2013) or in cis (Senner et al., 2011). The lncRNA locus (pink) may also encompass transcription factor binding sites (TF) that regulate the transcription of neighbouring genes. This effect may either be entirely independent of the lncRNA, or the binding of transcription factors may be affected positively or negatively by the act of transcription through the lncRNA locus. In this case, the mature RNA product would be incidental.

https://doi.org/10.7554/eLife.03058.003
Different strategies for analysis of lncRNA loss-of-function. Strategies that have been used to alter lncRNA function are described pictorially, with the wild type situation on the top-most line.

The lncRNA locus is indicated in pink, neighbouring protein-coding gene in blue, transcription factor binding sites within it by blue and purple ovals, transcriptional terminator sequences in yellow (‘Term’) and the process of transcription by grey dotted lines. Antisense oligonucleotides are able to bind to nascent RNA transcripts and trigger RNase H mediated degradation of the transcript in the nucleus. RNAi is elicited by short RNA species that bind to argonaute proteins (Ago, green oval) within the cell. This complex recognises complementary lncRNA molecules in the cytoplasm, and triggers their destabilisation by the endogenous cellular machinery. The CRISPR and TALE systems use designer DNA binding factors to recruit repressor or activator domains (orange oval) to the lncRNA to affect transcriptional initiation. The effects of each strategy upon the process of transcription and presence of underlying DNA elements such as transcription factor binding sites are indicated. The possibility of generating stable transgenic animals to investigate phenotypes throughout development is also noted.

https://doi.org/10.7554/eLife.03058.004
Human and mouse ENCODE data indicate that Mdgt−/− lines contain deletions of conserved binding sites for transcription factors and chromatin regulatory proteins.

The engineered deletion in mouse, and its equivalent sequence in human, are indicated by red rectangles, and spans 85% (12.4 kb of 14.7 kb) of intergenic sequence between mouse Hoxd1 and Hoxd3. Mdgt, virtually shares its start site with Hoxd1, a gene expressed with exquisite specificities in only a few cell populations during early development (Zakany et al., 2001). Predicted transcription factor binding sites (TFBs) that are conserved in human, mouse and rat are shown against the human genome (Consortium, 2012; Ernst and Kellis, 2012). Numbers of experimentally-determined TFBs per genomic interval are shown in the histogram, and clusters of DNase 1 hypersensitivity sites, are also shown aligned against the human locus. Predicted CpG islands acquired from the UCSC Genome Browser are shown in green, and chained human-mouse alignments are shown in olive green. Evolutionary conservation (GERP) scores are indicated below the mouse locus.

https://doi.org/10.7554/eLife.03058.005

Tables

Table 1

Representative studies that have disrupted lncRNA loci in vivo (N/A—not applicable)

https://doi.org/10.7554/eLife.03058.002
lncRNA nameOrganismMutation strategyReported animal phenotypeRNA-based rescue?Reference
XistMus musculus∼15 kb replaced with a neo expression cassetteFemales inheriting paternal allele were embryonic lethal; males fully viableNo(Marahrens et al., 1997)
XistMus musculusInversion of Exon 1 to intron 5Embryonic lethality of paternally inherited alleleNo(Senner et al., 2011)
H19Mus musculusReplacement by neo cassetteSlightly increased growthNo(Ripoche et al., 1997)
roXDrosophila melanogasterDeletions of roX1 or roX2None, except when in combination: male‐specific reduction in viabilityYes(Meller and Rattner, 2002)
Kcnq1ot1Mus musculusPromoter deletionGrowth deficiency for paternally inherited mutationNo(Fitzpatrick et al., 2002)
AirnMus musculusPremature transcriptional terminationGrowth deficiency for paternally inherited mutationNo(Sleutels et al., 2002)
Evf2Mus musculusPremature transcriptional terminationNoneN/A(Bond et al., 2009)
BC1Mus musculusReplacement of promoter and exon by PgkNeo cassetteVulnerable to epileptic fits after auditory stimulationNo(Zhong et al., 2009)
Neat1Mus musculus3 kb Promoter and 5’ deletionNoneN/A(Nakagawa et al., 2011)
TsxMus musculus2 kb Promoter and exon 1 deletionSmaller testes and less fearful (males)No(Anguera et al., 2011)
Malat1Mus musculusDeletionNoneN/A(Eissmann et al., 2012)
Malat1Mus musculuslacZ insertion and premature transcriptional terminationNoneN/A(Nakagawa et al., 2012)
Malat1Mus musculus3 kb Promoter and 5’ deletionNoneN/A(Zhang et al., 2012)
HotairMus musculusDeletionSpine and wrist malformationsNo(Li et al., 2013)
Hotdog and Twin of HotdogMus musculusLarge (28 Mb) translocation by inversionLoss of Hoxd expression in the cecumN/A(Delpretti et al., 2013)
FendrrMus musculusReplacement of exon 1 with transcriptional stop signalEmbryonic lethal around E13.75Yes (majority of embryos)(Grote et al., 2013)
FendrrMus musculusLocus replacement with lacZ cassettePerinatal lethalityNo(Sauvageau et al., 2013)
PerilMus musculusLocus replacement with lacZ cassettePerinatal lethalityNo(Sauvageau et al., 2013)
MdgtMus musculusLocus replacement with lacZ cassetteReduced viability and reduced growthNo(Sauvageau et al., 2013)
15 other lncRNA lociMus musculusLocus replacement with lacZ cassetteNoneN/A(Sauvageau et al., 2013)

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  1. Andrew R Bassett
  2. Asifa Akhtar
  3. Denise P Barlow
  4. Adrian P Bird
  5. Neil Brockdorff
  6. Denis Duboule
  7. Anne Ephrussi
  8. Anne C Ferguson-Smith
  9. Thomas R Gingeras
  10. Wilfried Haerty
  11. Douglas R Higgs
  12. Eric A Miska
  13. Chris P Ponting
(2014)
Science Forum: Considerations when investigating lncRNA function in vivo
eLife 3:e03058.
https://doi.org/10.7554/eLife.03058