#2 Story of 2021: Caltech Finds Amazing Role for Noncoding DNA
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The following was originally published on December 3, 2021.
Scientists at Caltech may have sounded the final death knell for the “junk DNA” myth. If only Dan Graur had known this years ago, it might have saved a lot of wasted rhetoric. ENCODE, readers recall, found that 80 percent of the genome is transcribed, even if only a small part codes for proteins. The functions of those non-coding regions were only hinted at. Now, the windows are opening on organization so all-encompassing for all those non-coding RNA transcripts, it is truly mind-boggling what goes on in the nucleus of a cell.
Using a new survey tool they call RD-SPRITE, Caltech researchers, in cooperation with others at USC and UCLA, mapped the spatial organization of all the DNA and RNA in the nucleus. It was challenging, they admit, to explore the spatial roles of RNA transcripts that don’t produce proteins, because the nucleus is a dynamic place crowded with DNA, proteins, and numerous RNAs of unknown function. Let them explain in their paper in Cell1 what they found in the resulting maps:
These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions. [Emphasis added.]
A new picture begins to emerge of hierarchical organization in the nucleus. No longer does it look like spaghetti in a basketball. On the contrary, there are territories and compartments throughout the interior. Caltech’s work adds to earlier knowledge of nuclear compartments:
- Nucleolus: contains transcribed ribosomal RNAs and associated processing molecules
- Speckles: contain pre-mRNAs and splicing components
- Transcriptional condensates: contain RNA polymerase II machines and factors
Now, additional hierarchical organization is being found in the nucleus. In the newly identified territories are found ncRNAs (noncoding RNAs, not destined for protein) — thousands of them — that are doing important jobs: guiding regulators into the right territories where “essential nuclear functions” need to take place.
Functional importance does not require that these ncRNAs exit the nucleus to be translated into proteins. Architecture is a function, is it not? Building designers carefully consider the floor plan of a large office, so that individuals can have cubicles for effective concentration as well as small conference rooms, large conference rooms, and labs for teamwork. What a picture is emerging of a fully functioning genome. It’s like a high-tech business organized for optimum workflow.
This is the first global map of nuclear organization that includes DNA, RNA and protein. Caltech scientists have been investigating how these easily diffusible molecules become spatially organized. Earlier, their SPRITE method, first reported in 2018, enabled them to examine pairwise contacts on a small scale. Now, their newly improved RD-SPRITE’s wide-angle mapping capability brings the overall organization into focus.
We recently developed SPRITE, which utilizes split-and-pool barcoding to generate comprehensive and multi-way 3D maps of the nucleus across a wide range of distances (Quinodoz et al., 2018). We showed that SPRITE accurately maps the spatial organization of DNA arranged around two nuclear bodies: nucleoli and nuclear speckles. However, our original version could not detect the majority of RNAs, including low abundance ncRNAs known to organize within several well-defined nuclear structures. Here, we introduce a dramatically improved method, RNA & DNA SPRITE (RD-SPRITE), which enables simultaneous, high-resolution mapping of thousands of RNAs, including low-abundance RNAs such as individual nascent pre-mRNAs and ncRNAs, relative to all other RNA and DNA molecules in 3D space. Using this approach, we identify several higher-order RNA-chromatin hubs and hundreds of ncRNAs that form high-concentration territories throughout the nucleus.
What happens in these territories? The paper focuses on some specific examples, showing that some of them combine the regulatory elements that facilitate and enhance the expression of genes and their mRNA transcripts. “Together, our results highlight a role for RNA [including non-coding RNA] in the formation of compartments involved in essential nuclear functionsincluding RNA processing, heterochromatin assembly, and gene regulation.”
Non-Coding RNA Importance
What had been previously referred to unflatteringly as “junk” turns out to be vital for efficient gene processing. Non-coding RNAs are transcripts of non-coding DNA (“junk DNA”). Here are specific examples of these ncRNAs at work.
- ncRNAs form “processing hubs” around genomic DNA regions that are being transcribed.
- ncRNAs and dozens of snoRNAs (small nucleolar RNAs) cluster around genes that are being transcribed for ribosomal RNAs. These ncRNAs “form multi-way contacts with each other,” they found.
- ncRNAs cluster at splicing sites where there is a high density of RNA polymerase II (Pol II, the transcription machinery).
- ncRNAs are associated closely with the formation of small nuclear RNAs (snRNAs). These snRNA transcripts are involved in “functional components of the spliceosome at thousands of nascent pre-mRNA targets.”
- ncRNAs concentrate around transcripts where centromeres are forming. “We found that these ncRNAs primarily localize over centromere-proximal regions… and organize into higher-order structures containing these ncRNAs and multiple centromere-proximal regions from different chromosomes.”
Associating together at these processing hubs, the ncRNAs and their ligands bring together components for interaction even over long genomic distances. “Despite being separated by large genomic distances, these DNA regions form long-range contacts,” the paper says.
Together, these results indicate that higher-order spatial organization of diffusible regulators around shared DNA sites and their corresponding nascent RNA targets is a common feature of many forms of RNA processing.
In each of these examples, we observed spatial compartments that consist of: (1) nascent RNAs localized near their DNA loci, (2) these DNA loci forming long-range 3D contacts, and (3) diffusible ncRNAs associating with these nascent RNAs and DNA loci within the compartment.
In short, “Hundreds of non-coding RNAs localize in spatial proximity to their transcriptional loci.” This also applies to long non-coding RNAs (lncRNAs). “These results demonstrate that many hundreds of lncRNAs form high-concentration spatial territories throughout the nucleus.”
The ncRNAs do more than just form compartments. They recruit proteins to come and join the army! “Because hundreds of lncRNAs are enriched in territories throughout the nucleus, we explored whether RNAs might impact protein localization within these territories,” they said on a hunch. Sure enough, additional experimentation found this to be the case.
These results demonstrate that the localization pattern of a lncRNA in 3D space can act to guide recruitment of regulatory proteins to specific nuclear territories and highlights an essential role for these lncRNA-enriched nuclear territories in gene regulation.
The roles of these transcripts, formerly thought to be useless, have risen to the status of essential players. What an amazing turnaround! The conclusion of this Caltech paper, which never mentions evolution, appears to nail the case shut for junk DNA, while also adding new levels of awe to the workings inside the nucleus.
Our results demonstrate that ncRNAs can act as seeds to drive spatial localization of otherwise diffusive ncRNA and protein molecules. We showed that experimental perturbations of several ncRNAs disrupt localization of diffusible proteins… in dozens of compartmentalized structures. In all cases, we observed a common theme where (1) specific RNAs localize at high concentrations in proximity to their transcriptional loci and (2) diffusible ncRNA and protein molecules that bind to them are enriched within these structures. Together, these observations suggest a common mechanism by which RNA can mediate nuclear compartmentalization: nuclear RNAs can form high-concentration spatial territories close to their transcriptional loci (‘‘seed’’), bind to diffusible regulatory ncRNAs and proteins through high-affinity interactions (‘‘bind’’), and thus act to dynamically change the distribution of diffusible molecules such that they become enriched within these territories (‘‘recruit’’; Figure 7). By recruiting diffusible regulatory factors to multiple DNA sites, these ncRNAs may also act to drive coalescence of distinct DNA regions into a shared territory in the nucleus. This may explain why various RNAs are critical for organizing long-range DNA interactions around specific nuclear bodies.
This is a paper to remember. It shows in hindsight the fruitfulness of the ID perspective over the evolutionary one. Evolutionary thinking dismissed these non-coding RNAs as junk. ID thinking would have approached the unknown with the premise, “If something works, it’s not happening by accident.”
- Quinodoz et al., RNA promotes the formation of spatial compartments in the nucleus. Cell, 4 Nov 2021, https://doi.org/10.1016/j.cell.2021.10.014.