Kotaro Nakanishi
Ohio State University, OH·Biochemistry
Cells constantly fine-tune which genes are active. One way is through microRNAs (miRNAs), RNA strands about 22 nucleotides long that switch off specific genes after they are copied into messenger RNA (mRNA).
- Fields
Biochemistry
Biophysics
Genetics
- Topics
microRNA
miRNA
small noncoding RNA
- Grants
- 3 known grants, through Jan 2031
What they work on
- Determine how AGO2 and AGO3 loaded with cleavage-inducing tiny RNAs (cityRNAs) recognize the sequence just upstream of their target site and cut target RNA, using structural studies and RNA sequencing.
- Solve cryo-EM or crystal structures of neurodevelopmental disorder-linked AGO1 and AGO2 mutants loaded with miRNAs or unusually short tyRNAs, to see how the mutations change Argonaute structure.
- Examine how AGO1 interacts with PARN, an RNA-trimming enzyme, to explain the abnormal miRNA trimming seen in disease-linked AGO1 mutants.
- Test whether disease-linked AGO2 mutants trim miRNAs like mutant AGO1 and whether their tyRNA-loaded complexes can still silence genes, and map where they sit relative to AGO-binding proteins in cells.
- Determine the structure of the PARN-AGO complex bound to a miniature mRNA to explain how PARN trims miRNAs to about 22 nucleotides and selectively degrades specific mRNAs.
- Capture cryo-EM structures of each step of RISC assembly to reveal how Argonaute picks the less tightly paired end of an RNA duplex to choose its guide strand.
- Compare target recognition by all four human Argonautes by solving cryo-EM and X-ray structures of each RISC with the same guide and target RNAs, and probe target RNA motion with a SHAPE-based chemical mapping method.
- Identify each AGO's unique protein partners with mass spectrometry, then use tandem immunoprecipitation and RNA sequencing to test how these partners shape which RNAs each AGO targets.
More about the research
A miRNA works only when loaded into an Argonaute (AGO) protein. The pair forms an RNA-induced silencing complex (RISC) that finds and silences matching target RNAs. Humans have four AGO proteins, long assumed to do the same job. This research uses protein structures and biochemistry to ask how each AGO recognizes its targets, how small RNAs are made and trimmed, and how AGO mutations lead to disease. The lab's structural studies have revealed differences among the four human AGOs. They also led to the discovery of tiny guide RNAs (tyRNAs), AGO-bound guides 17 nucleotides or shorter. The team found that specific exonucleases, enzymes that chew RNA from one end, trim 21–23-nucleotide miRNAs into 14-nucleotide tyRNAs. Some of these, called cleavage-inducing tyRNAs (cityRNAs), switch on AGO3's ability to cut RNA. Their work showed that cityRNAs silence genes mainly by cutting target RNAs rather than by blocking translation or destabilizing mRNA.
Why it matters
RNA interference (RNAi) drugs rely on small guide RNAs loaded into Argonaute proteins. Knowing exactly how each human AGO picks and cuts its targets could help design more accurate guide-RNA therapeutics and better target-prediction tools. Explaining how AGO1 and AGO2 mutations produce abnormal tiny RNAs may clarify the molecular basis of Argonaute-linked neurodevelopmental disorders. It could also inform future approaches to microRNA-related diseases.
Known grants
From NIH and NSF research grants. Labs often have other funding we can't see.
Closest labs
Phillip Zamore
University of Massachusetts Chan Medical School, MA·Biochemistry
Identify the regulatory targets and gene-control mechanisms of pachytene piRNAs, small RNAs abundant in mouse sperm-precursor cells, using biochemistry and mouse genetics.
Clara Kielkopf
University of Rochester, NY·Biochemistry
Compare how recurrent U2AF2 mutations found in neurodevelopmental disorders and in cancers affect RNA binding and splicing, using structural methods and pre-mRNA splicing assays in human cells.
Scott Bailey
Johns Hopkins University, MD·Biochemistry
Determine how CRISPR-associated proteins use small guide RNAs to locate and destroy foreign DNA or RNA in prokaryotes, using cryo-EM structures together with biochemical, biophysical and cell-based assays.
The school
Ohio State University: 71 labs on PI Pages, $1.6B a year on research, 882 research doctorates a year.
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