ASAR lncRNAs control DNA replication timing through interactions with multiple hnRNP/RNA binding proteins
Curation statements for this article:-
Curated by eLife
eLife assessment
The findings in this manuscript are important and novel, and the genomic analyses are convincing. This study expands upon our understanding of the role of hnRNP proteins in lncRNA function and the evidence is compelling, suggesting shared mechanism(s) in the regulation of ASARs and Xist RNAs by RBPs that bind Cot1 sequences in these lncRNAs. This manuscript should be of interest to the noncoding RNA and chromatin biology communities.
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Abstract
ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An ∼7kb domain within the ASAR6-141 lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ∼7kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes in cis . shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.
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eLife assessment
The findings in this manuscript are important and novel, and the genomic analyses are convincing. This study expands upon our understanding of the role of hnRNP proteins in lncRNA function and the evidence is compelling, suggesting shared mechanism(s) in the regulation of ASARs and Xist RNAs by RBPs that bind Cot1 sequences in these lncRNAs. This manuscript should be of interest to the noncoding RNA and chromatin biology communities.
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Reviewer #1 (Public Review):
Summary:
Thayer et al build upon their prior findings that ASAR long noncoding RNAs (lncRNAs) are chromatin-associated and are implicated in control of replication timing. To explore the mechanism of function of ASAR transcripts, they leveraged the ENCODE RNA binding protein eCLIP datasets to show that a 7kb region of ASAR6-141 is bound by multiple hnRNP proteins. Deletion of this 7kb region resulted in delayed chromosome 6 replication. Furthermore, ectopic integration of the ASAR6-141 7kb region into autosomes or the inactive X-chromosome also resulted in delayed chromosome replication. They then use RNA FISH experiments to show that the knockdown of these hnRNP proteins disrupts ASAR6-141 localization to chromatin and in turn replication timing.
Strengths:
Given prior publications showing HNRNPU to be …
Reviewer #1 (Public Review):
Summary:
Thayer et al build upon their prior findings that ASAR long noncoding RNAs (lncRNAs) are chromatin-associated and are implicated in control of replication timing. To explore the mechanism of function of ASAR transcripts, they leveraged the ENCODE RNA binding protein eCLIP datasets to show that a 7kb region of ASAR6-141 is bound by multiple hnRNP proteins. Deletion of this 7kb region resulted in delayed chromosome 6 replication. Furthermore, ectopic integration of the ASAR6-141 7kb region into autosomes or the inactive X-chromosome also resulted in delayed chromosome replication. They then use RNA FISH experiments to show that the knockdown of these hnRNP proteins disrupts ASAR6-141 localization to chromatin and in turn replication timing.
Strengths:
Given prior publications showing HNRNPU to be important for chromatin retention of XIST and Firre, this work expands upon our understanding of the role of hnRNP proteins in lncRNA function.
Weaknesses:
The work presented is mechanistically interesting, however, one must be careful with the over-interpretation that hnRNP proteins can regular chromosome replication directly. Furthermore, the work could be strengthened by including a few controls and clarifications.
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Reviewer #2 (Public Review):
Summary:
This paper reports a role for a substantial number of RNA binding proteins (RBPs), in particular hnRNPs, in the function of ASAR "genes". ASARs are (very) long, non-coding RNAs (lncRNAs) that control allelic expression imbalance (e.g.: mono-allelic expression) and replication timing of their resident chromosomes. These relatively novel "genes" have recently been identified on all human autosomes and are of broad significance given their critical importance for basic chromosomal functions and stability. However, the mechanism(s) of ASAR function remain unclear. ASARs exhibit some functional relatedness to Xist RNA, including persistent association of the expressed RNA with its resident chromosome, and similarities in the composition of RNA sequences associated with ASARs, in particular Line1 RNAs. …
Reviewer #2 (Public Review):
Summary:
This paper reports a role for a substantial number of RNA binding proteins (RBPs), in particular hnRNPs, in the function of ASAR "genes". ASARs are (very) long, non-coding RNAs (lncRNAs) that control allelic expression imbalance (e.g.: mono-allelic expression) and replication timing of their resident chromosomes. These relatively novel "genes" have recently been identified on all human autosomes and are of broad significance given their critical importance for basic chromosomal functions and stability. However, the mechanism(s) of ASAR function remain unclear. ASARs exhibit some functional relatedness to Xist RNA, including persistent association of the expressed RNA with its resident chromosome, and similarities in the composition of RNA sequences associated with ASARs, in particular Line1 RNAs. Recent findings that certain hnRNPs control the chromosome territory retention of Cot1-bearing RNAs (which includes Line1) led the authors to test the hypothesis that hnRNPs might regulate ASARs.
Specific new findings in this paper:
-Analysis of eCLIP (RNA-protein interaction) ENCODE data shows numerous interactions of the ASAR6-141 RNA with RBPs, including hnRNPs (e.g.: HNRNPU) that have been implicated in the retention of RNAs within local chromosome territories.
-Most of these interactions can be mapped to a 7kb region of the 185kb ASAR6-141 RNA.
-Deletion of this 7kb region is sufficient to induce the DMC/DRT phenotype associated with deletion of the entire ASAR region.
-Ectopic integration into mouse autosomes of the 7kb region is sufficient to cause DMC/DRT of the targeted autosome, and a similar effect upon ectopic integration into inactive X. This raises the question about integration into the active X, which was not mentioned. Is integration into the active X observed? Is it possible that integration might alter Xist expression confounding this interpretation?
-Knockdown of RBPs that bind the 7kb region causes dissociation of ASAR6-141 RNA from its chromosome territory, and, remarkably, dissociation of Xist RNA from inactive X, and mis-colocalization of the ASAR6-141 and Xist RNAs. Depletion of these RBPs causes DMC/DRT on all autosomes.
Strengths:
These are compelling results suggesting shared mechanism(s) in the regulation of ASARs and Xist RNAs by RBPs that bind Cot1 sequences in these lncRNAs. The identification of these RBPs as shared effectors of ASARs and Xist that are required for RNA territory localization mechanistically links previously independent phenomena.
The data are convincing and support the conclusions. The replication timing method is low resolution and is only a relative measure but seems adequate for the task at hand. The FISH experiments are convincing. The quality of the images is impressive.
Links to other subfields like X-inactivation and RNA association with chromosome territories provide novel context and protein players, new phenotypes to examine.
Weaknesses:
The exact effects of knockdown experiments are unclear and may be indirect, which is acknowledged.
The mechanism is not much clearer than before.
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