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Sequencing Studies Reveal New RNA Secondary Structure Details

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Sequencing-based strategies are at the heart of several recent studies by researchers set on surveying RNA secondary structure profiles in a given organism or cell type, adding to the structural data that can be obtained by more targeted but time-consuming methods.

In the January 30thissue of Nature, a trio of independent research teams assessing RNA secondary structural patterns in various cell types in vitro or in vivo turned to sequencing to profile RNA transcripts that had been treated with either dimethyl sulfate (DMS) — a compound that methylates adenine and cytosine bases in single-stranded RNA — or with a pair of structure-specific nuclease enzymes.

Using a DMS-based "Structure-seq" scheme, for instance, investigators from Pennsylvania State University interrogated secondary structures of RNA in vivo in Arabidopsis thaliana seedlings. That study pointed to a three-nucleotide periodicity in RNA structure across coding transcripts, consistent with prior studies in yeast cells profiled in vitro.

The study's authors also detected structural differences that appear to be related to the biological roles of some mRNAs, such as transcripts from stress response genes.

A University of California at San Francisco-led team used a similar DMS-based sequencing strategy to compare in vitro and in vivo RNA secondary structures in yeast and mammalian cell types. That analysis revealed a range of RNA structure differences in cells interrogated in vivo and in vitro, including a dip in structured mRNAs in vivo in quickly dividing yeast cells.

The group also identified parts of the transcriptome with very distinct structural profiles in the in vivo and in vitro experiments, in part due to active unwinding processes at play in vivo.

"Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure," UCSF cellular and molecular pharmacology researcher Jonathan Weissman, the study's corresponding author, and his colleagues wrote.

"Indeed," they added, "analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells."

A team from the US, Israel, and Singapore used a distinct transcriptome treatment for their sequencing study of RNA secondary structures in human lymphoblastoid cells from multiple members of the same family.

That group did deep sequencing on bits of RNA generated with the RNase V1 enzyme, which cuts double-stranded RNA, or with the S1 nuclease, which preferentially dices up single-stranded RNA.

Relying primarily on that parallel analysis of RNA structure (PARS) sequencing approach — introduced in a 2010 Nature study of budding yeast mRNA structure by members of the same team — the researchers assessed the secondary structures of both coding and non-coding transcripts from members of a parent-child trio.

In the process, they not only identified RNA secondary structure, or RSS, signatures present in different transcripts types, but also explored the interplay between RSS and sequence variation. The latter analysis turned up RSS-related roles for some 15 percent of the 12,200 or so transcribed single nucleotide variants considered, suggesting that a subset of "riboSNitches" may also influence human physical features or disease risk states.

Such features are important, the researchers explained, because the conformation on RNA transcripts in a given cell is intimately linked to the way that genes are ultimately expressed as proteins.

There are several strategies for assessing RSS that don't involve high-throughput sequencing, noted that study's co-first author Yue Wan, a stem cell and development researcher affiliated with the Genome Institute of Singapore and Stanford University, including schemes based on gel electrophoresis or capillary sequencing of chemically or enzymatically probed RNAs.

But most provide structural information on just a few hundred RNA bases at a go, Wan told In Sequence in an email message, noting that deep sequencing significantly ramps up that throughput, making it possible to do structure probing on thousands of RNAs simultaneously.

Amongst the chemical probing that have been combined with deep sequencing to distinguish RNA transcripts with different structural features are selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) and DMS treatment.

The latter approach methylates adenine and cytosine bases present in single-stranded transcripts, Wan said, explaining that the "readout for the methylated bases is through reverse transcriptase stoppages."

"As reverse transcriptase stoppages can also occur at other regions along the RNA, the background noise of sequencing using chemical probes is high," she said.

For their part, she and her colleagues have been performing RNA secondary structure, or RSS, analyses using PARS — a method that uses RNase V1 and S1 nuclease enzymes that cut RNA differently depending on its structural context.

After isolating polyadenylated transcripts from a given sample, the team treats parallel samples with the double-stranded RNA cutting RNase V1 enzyme and the single-stranded RNA cleaver, S1.

That exposes residues that can be bound by 5' adaptors prior to the reverse transcription, amplification, and deep sequencing steps, she explained. And by looking at which bases appear directly after the adaptor, it's possible to see the site where single- or double-stranded enzyme cutting occurred.

So far Wan and her co-authors have successfully applied the PARS approach using Illumina and SOLiD platforms, in each case generating data that provide a fairly consistent picture of RSS. In theory, though, the same approach should be compatible with any sequencing instrument that relies on ligation-based library preparation.

The approach required new analytical methods for not only combining information from the double- and single-stranded transcript reads to determine which RNA bases were paired and which were not, but also to untangle variant-associated structural changes in the transcripts.

In their Nature study, the researchers' PARS-based look at RSS pointed to RSS signatures associated with open reading frames and with splice junctions where different expressed sequences, or exons, bump up against one another, for example, pointing to an apparent role for RNA splicing in RSS.

"We observed a distinct RSS around RNA exon-exon junctions," Wan said, noting that more research is needed to understand how splicing regulates RSS, and vice versa.

But the RSS information was not limited to protein-coding transcripts. On the contrary, their analysis highlighted sequence features defining binding sites in microRNAs and other non-coding transcript types as well.

It also indicated that, in contrast to patterns detected in yeast cells, the secondary structures of human RNAs were more pronounced in untranslated regions rather than coding regions of transcripts.

That study described largely comparable RSS patterns from in vitro experiments and experiments on human RNAs isolated from human cells using methods designed to reduce RNA structure disruption, Wan noted.

Even so, the group did see a fraction of transcriptome regions with significantly different structures in vitro, consistent with some of the findings described in the comparison study by Weissman and colleagues.

Using the data on lymphoblastoid cells from a child and both of his or her parents, meanwhile, the team also took a closer look at the consequences of genetic variation in this process, Wan noted.

"As the mutations between the father and the mother are inherited in the child," she said, "the structural changes between the [two] individuals can be further validated in their offspring, enabling us to validate the structural changes that we observed in the parents."

In particular, they uncovered more than 1,900 SNVs that seem to alter the local secondary structure of the RNA transcripts containing them. That's far more than anticipated based on past studies, Wan noted, and hints at a pronounced role for SNVs in governing RNA structure and related functional processes.

"[T]he landscape and variation of RSS across human transcriptomes suggest important roles of RNA structure in many aspects of gene regulation," Wan and co-authors concluded.

As they continue exploring such features and their effects, the researchers are interested in using in vivo strategies to look at how variant-associated RSS alterations impact cells' structural architecture, for example, and ways in which such structural changes shift in disease states compared to healthy samples from the same tissue.

"More work needs to be performed to understand mechanistically how these structure-changing mutations result in functional consequences in the cell," Wan said.

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