RNA structures use and depend on the material within living cells, but how, exactly, is an unanswered question. A new tool may offer insight into where the loops and hairpins on viral RNA exist and how they interact with RNA in their hosts, helping to shed light on viruses ranging from Zika to SARS-CoV-2, which causes COVID-19.

NIEHS Distinguished Lecture Series speaker Eric Miska, Ph.D., from the University of Cambridge, has studied RNA communication, how certain chromosomal segments shape fungal evolution, and how environmental factors affect gene plasticity, adaptation, and inheritance. His lab’s research into a new method of looking at RNA structures could help us better understand the genomics of viruses, he told a virtual audience on May 10.

Miska has made “critical contributions to the field of epigenetic changes guided by small RNAs,” said event host Marcos Morgan, Ph.D., a Stadtman Investigator in the Male Reproduction and RNA Biology Group. “He discovered the multi-generational transmission of epigenetic memory through a type of small RNA in worms. More recently, he continued this line of research on mammalian systems, investigating how epigenetic changes induced by stress can be transmitted through sperm RNA to the next generation.”

Sleuthing structures

Omar Ziv, Ph.D., while a research fellow in Miska’s lab, developed a method called cross-linking of matched RNAs and deep sequencing (COMRADES) to detect RNA structures. The research team used modified psoralen, a molecule long studied by RNA biologists that can travel easily across cell membranes, in a live cell line to look for areas where RNA exhibits double-stranded structures. They then purified a selection of RNA and the crosslinked areas of interest.

“That’s the special trick that Omar developed and optimized,” said Miska. “Purifying crosslinks gives us about a million-fold enrichment of these over other bits of RNA. And this is really where the power of this very simple method lies.”

RNA that contain crosslinks are glued together and sequenced. When the researchers tested the method on human ribosomal RNA, they found it was highly reproducible and effective, detecting more than 90% of the base pairs.

Untangling viral RNA knots

To test the method further, the team focused on RNA viruses, which are highly structured with variable shapes. Their work centered on flaviviruses that include Zika, dengue, West Nile, and yellow fever.

Roughly 5% of the Zika virus structure is known, prompting the researchers to use the COMRADES method to investigate the other 95% and look for any host-virus RNA interactions. They found they could detect all the known structural features of Zika, including the hairpins and knots in the RNA.

Dynamic depending on environment

Yet RNA structures within a cell are dynamic living things that might change, Miska said.

Two different shapes on RNA represent the two main functions of the viral genome: to translate and to replicate. When the scientists looked at the Zika virus in two hosts, a human placental cell line and an insect cell line, the team found that the structures of the virus in both lines were similar despite differences in temperature and general environments.

However, there are certain structural features that are completely and reproducibly different depending on what host cell the virus invades.

Slowing down Zika

In the human cell line, the team detected that the Zika virus interacts with miR-21, an abundant microRNA. MiR-21 interacts in the position where the Zika virus RNA comes together to replicate the enzyme creatine kinase, which is found in skeletal and heart muscle and the brain. If the Zika virus base pairing is changed, the miR-21 will not bind to the viral RNA, showing the team that efficient RNA replication requires this microRNA.

Using CRISPR, a technology to edit genes (see sidebar), the team knocked out miR-21 in host cells to see how it would affect the Zika virus, finding that it reduced the rate of the viral RNA replication by 50%. They got the same result by introducing a miR-21 inhibitor, suggesting that miR-21 is important to the virus.

The lab then teamed up with collaborators to use COMRADES on SARS-CoV-2, the largest RNA genome in nature and the virus that causes COVID-19. Within it, they found long-range interactions, including a huge loop along a particular stretch of RNA. The potential for COMRADES is vast, Miska indicated, and it could detect other structures in viral RNA that researchers could eventually target in future studies.

Citation: Ziv O, Gabryelska MM, Lun ATL, Gebert LFR, Sheu-Gruttadauria J, Meredith LW, Liu ZY, Kwok CK, Qin CF, MacRae IJ, Goodfellow I, Marioni JC, Kudla G, Miska EA.. 2018. COMRADES determines in vivo RNA structures and interactions. Nat Methods. 15(10):785–788.

(Susan Cosier is a contract writer for the NIEHS Office of Communications and Public Liaison.)