RNA structures use and depend on material in living cells, but how, exactly, is an unanswered question. A new tool may offer insight into where loops and hairpins are on viral RNA and how they interact with their hosts’ RNA, 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, studied RNA communication, how certain chromosomal segments shape fungal evolution, and how environmental factors affect gene plasticity, adaptation, and inheritance. His lab’s research on a new way to look at RNA structures could help us better understand the genomics of viruses, he told a virtual audience on May 10.
Miska has made “essential contributions to the field of small RNA-guided epigenetic changes,” said event host Marcos Morgan, Ph.D., a Stadtman researcher in the Male Reproduction and Biology Group at the ‘RNA. “He discovered the multigenerational transmission of epigenetic memory through a type of small RNA in worms. More recently, he has continued this line of research in mammalian systems, investigating how stress-induced epigenetic changes can be transmitted through sperm RNA to the next generation.
Omar Ziv, Ph.D., while a researcher in Miska’s lab, developed a method called cross-linking 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 move easily across cell membranes, in a living cell line to look for areas where the RNA exhibits structures double stranded. They then purified a selection of RNA and cross-linked areas of interest.
“That’s the special trick that Omar developed and optimized,” Miska said. “Purifying the crosslinks gives us about a million-fold enrichment compared to other pieces of RNA. And that’s really where the power of this very simple method lies.
RNAs that contain crosslinks are glued together and sequenced. When researchers tested the method on human ribosomal RNA, they found it to be highly reproducible and efficient, detecting over 90% of base pairs.
Untangling viral RNA knots
To further test the method, the team focused on RNA viruses, which are highly structured with variable shapes. Their work has focused on flaviviruses which include Zika, Dengue, West Nile and Yellow Fever.
About 5% of the structure of the Zika virus is known, prompting researchers to use the COMRADES method to study the remaining 95% and look for any host-virus RNA interactions. They found they could detect all known structural features of Zika, including hairpins and knots in RNA.
Dynamics depending on the environment
Yet RNA structures within a cell are dynamic living things that could change, Miska said.
Two different shapes on the RNA represent the two main functions of the viral genome: to translate and to replicate. When scientists examined 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 the two lineages were similar despite differences in temperature and environment. general.
However, some structural features are completely and reproducibly different depending on the host cell invaded by the virus.
Slow 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 Zika virus RNA gathers to replicate the enzyme creatine kinase, which is found in skeletal and cardiac muscle and the brain. If Zika virus base pairing is altered, miR-21 will not bind to viral RNA, showing the team that efficient RNA replication requires this microRNA.
Using CRISPR, a technology to edit genes (see box), 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 viral RNA replication by 50%. They obtained the same result by introducing an inhibitor of miR-21, suggesting that miR-21 is important for 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. Inside, they found long-range interactions, including a huge loop along a particular stretch of RNA. The potential for COMRADES is vast, Miska said, and it could detect other structures in viral RNA that researchers could potentially target in future studies.
Quote: 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 RNA structures and interactions in vivo. National methods. 15(10):785–788.
(Susan Cozier is a contract writer for the NIEHS Office of Communications and Public Liaison.)