Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression
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eLife assessment
This study concerns an important area, that of monoallelic expression, but the study does not provide sufficient information about the candidate regulatory RNA to provide a significant advance over previous work, which should also be discussed in more detail.
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Abstract
Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.
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Author Response
Joint Public Review:
These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript.
We apologize for not including the required details in the manuscript. The cell cycle lncRNA screen where we identified the initial SNUL-1 probe was published in an earlier paper 6. By performing RNA-seq in cell cycle synchronized samples, we identified several hundreds of lncRNAs that differentially expressed in a particular stage of the cell cycle. We performed a large-scale RNA-FISH-based screen to characterize the localization of these cell cycle-regulated lncRNAs. One of the probes in this screen hybridized to SNUL-1 RNA in the nucleolus. The original double-stranded DNA probe that detected the …
Author Response
Joint Public Review:
These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript.
We apologize for not including the required details in the manuscript. The cell cycle lncRNA screen where we identified the initial SNUL-1 probe was published in an earlier paper 6. By performing RNA-seq in cell cycle synchronized samples, we identified several hundreds of lncRNAs that differentially expressed in a particular stage of the cell cycle. We performed a large-scale RNA-FISH-based screen to characterize the localization of these cell cycle-regulated lncRNAs. One of the probes in this screen hybridized to SNUL-1 RNA in the nucleolus. The original double-stranded DNA probe that detected the SNUL-1 RNA cloud(s) was mapped to hg38-Chr17: 39549507-39550130 genomic region, encoding a lncRNA. However, other unique non-overlapping probes generated from the Chr17-encoded lncRNA failed to detect the SNUL-1 RNA cloud. Furthermore, BLAST-based analyses failed to align the SNUL-1 hybridized sequence to any other genomic loci. Since a large proportion of the p-arms of nucleolus-associated NOR-containing acrocentric chromosomes is not yet annotated, we speculated that SNUL-1 could be transcribed from an unannotated genomic region from the acrocentric p-arms.
We have now provided the information in the revised manuscript. Specifically, we have provided the details of the PacBio iso-seq, nanopore seq analyses as well as the bioinformatic approaches that were conducted to determine the identity of the full-length SNUL-1 ncRNA.
If these are RNAs with reasonable abundance, then they should be findable without the extensive PCR amplification they appear to have done for the PacBio sequencing (the methods section is not clear on exactly how many rounds of PCR were performed).
We apologize for not providing the essential details. In the PacBio-iso-seq analyses, we utilized the standard protocol (recommended by the scientists from PacBio, who are authors in the manuscript), which included 13 PCR cycles. However, as described in the manuscript, in parallel to PacBio-seq, we also performed nano-pore sequencing of the nucleolus-enriched RNA without any amplification. The SNUL-1 full-length candidate sequence (CS) that we described in the manuscript is the ncRNA that showed 100% sequence similarity in both independent PacBio Iso-seq as well as nanopore seq analyses. We argue that if the SNUL-1 candidate transcripts would have been an artifact of PCR amplification in PacBio-seq, then we would not have obtained the full-length sequence with 100% match in the nanopore-seq reads. We have now included the detailed bioinformatic analyses in the methods section of the ms.
Moreover, given the acknowledged sequence similarities of the SNULs with other RNAs, the possibility of chimaera formation during PCR amplification is high. They are clearly detecting RNAs associated with nucleoli but exactly what they are examining is unclear.
Please see our response above (public Reviewer comment 2). In addition, we performed detailed bioinformatic analyses to test whether the SNUL-1 full-length sequence obtained in the PacBio-seq is not an artifact of PCR amplification. This analysis is described in detail in the methods section under the sub-title “sequencing analyses”.
It is possible that a clear determination of the genomic origin of these RNAs will be complicated by the repetitive sequences in the regions of the genome where they reside.
We thank this reviewer for acknowledging the technical limitation of mapping the genomic locus of SNUL1 genes. We have pointed out this as the limitation of the present manuscript. Mapping the SNUL-1 genomic locus and characterizing the regulatory sequence elements and factors that control the monoallelic expression of SNULs will be part of future research plans.
Note also that the idea of monoallelic expression from rRNA encoding loci is interesting, but has been established in 2009. Title: Allelic inactivation of rDNA loci. Genes Dev. 2009 Oct 15;23(20):2437-47. doi: 10.1101/gad.544509.
We thank the reviewer for pointing out the study from Cedar lab published in 2009. To test the idea that SNULs contribute to allele specific expression of rRNA, which was previously reported by Cedar lab in their 2009 G&D paper, we performed the same set of experiments described in their paper in three different cell lines in the presence or absence of SNULs (please see the response to Editorial comment-2). However, we could not reproduce any of the data presented in the G&D manuscript. Also, we have not seen any other follow up study, where mono-allelic expression of rDNA genes was observed. Currently, no concrete data supports monoallelic expression of rRNA 5. We, therefore, argue that our current study is the first one, demonstrating the mono-allelic association of a ncRNA from the p-arm containing rDNA cluster.
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eLife assessment
This study concerns an important area, that of monoallelic expression, but the study does not provide sufficient information about the candidate regulatory RNA to provide a significant advance over previous work, which should also be discussed in more detail.
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Joint Public Review:
The manuscript "Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression" reports the discovery of a family of ncRNAs they call SNULs for Single NUcleolus Localized RNA and examine their localization with respect to nucleoli and reports that the RNAs they are examining are monoallelically expressed in a mitotically stable manner similar to what happens in X inactivation.
These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript. If these are RNAs with reasonable abundance, then they should be findable without the extensive PCR amplification they appear to have done for the PacBio sequencing (the methods section is …
Joint Public Review:
The manuscript "Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression" reports the discovery of a family of ncRNAs they call SNULs for Single NUcleolus Localized RNA and examine their localization with respect to nucleoli and reports that the RNAs they are examining are monoallelically expressed in a mitotically stable manner similar to what happens in X inactivation.
These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript. If these are RNAs with reasonable abundance, then they should be findable without the extensive PCR amplification they appear to have done for the PacBio sequencing (the methods section is not clear on exactly how many rounds of PCR were performed). Moreover, given the acknowledged sequence similarities of the SNULs with other RNAs, the possibility of chimaera formation during PCR amplification is high. They are clearly detecting RNAs associated with nucleoli but exactly what they are examining is unclear. It is possible that a clear determination of the genomic origin of these RNAs will be complicated by the repetitive sequences in the regions of the genome where they reside.
Note also that the idea of monoallelic expression from rRNA encoding loci is interesting, but has been established in 2009. Title: Allelic inactivation of rDNA loci. Genes Dev. 2009 Oct 15;23(20):2437-47. doi: 10.1101/gad.544509.
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