Protein levels in mammalian cells are set largely by the abundance of their messenger RNAs (mRNAs). The mRNA abundance depends on mRNA production and decay, which change according to the needs of the cell. The production is controlled by transcription factors or by chromatin modifications epigenetically, whereas decay is controlled by microRNAs (miRNAs), by proteins that bind to AU-rich RNA elements (AREs) in mRNAs, as well as by poly-U tail addition or m6A methylation.
In addition, mammalian cells contain a variety of sensors that survey conditions inside the cells (for example, DNA damage, viral or bacterial molecules) or outside the cells (for example, cytokines, chemokines, interferons, viral or bacterial molecules), and tune the levels of mRNAs involved in immune and stress programs. These programs either help the cell to adapt to the damage or kill the cell to benefit survival of the whole organism. They are important not only for normal cellular functions, but also for infectious and neoplastic diseases, inflammatory and degenerative disorders, diabetes and innate (i.e. pre-programmed) immunity.
We are interested in these mechanisms.
Receptors of dsRNA. Our work is providing structural and biochemical insights into double-stranded RNA (dsRNA) sensors, which activate mRNA decay by producing a signaling RNA-like molecule "2-5A" with unusual 2',5'-linkages. The dsRNAs molecules are not abundant in normal cells, but they become abundant upon viral infections or DNA damage that presumably strips off repressive DNA methylation marks from endogenous retrovirus sites. The endogenous dsRNA production, surveillance and physiologic roles are very intriguing and not fully understood.
STRUCTURE OF HUMAN OAS1 WITH dsRNA
Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1.
HUMAN OAS3 IS A SENSOR OF LONG dsRNA
Human cells express three related but different sensors of dsRNA that synthesize 2-5A. Using X-ray crystallography and biochemistry we determined that the largest OAS family member, OAS3, is a selective sensor of long dsRNA. OAS3 contains three duplicated domains. The C-terminal domain (DIII) is catalytically active and makes 2-5A, whereas the N-terminal domain (DI) is catalytically inactive and serves for high-affinity dsRNA recruitment and for measurement of minimal dsRNA length.
Structural mechanism of sensing long dsRNA via a noncatalytic domain in human oligoadenylate synthetase 3.
DsRNA-induced mRNA decay. We are also learning about the 2-5A receptor, RNase L. RNase L contains a protein kinase domain and belongs to the family of more than 500 mammalian protein kinases. RNase L does not phosphorylate any known proteins, which makes it different from most kinases. Uniquely, RNase L cleaves cellular RNAs during the interferon response, which protects the cells from viruses and bacteria, and blocks cell proliferation. We solved the crystal structure of human RNase L with bound 2-5A and RNA substrate, which explains how this enzyme is regulated.
STRUCTURE OF HUMAN RNASE L WITH 2-5A AND RNA SUBSTRATE
Structure of human RNase L reveals the basis for regulated RNA decay in the IFN response.
Innate immune messenger 2-5A tethers human RNase L into active high-order complexes.
Leading Edge Select: The Fantastic RNase L
Another area of our work is understanding the precise mechanism and roles of mRNA cleavage by RNase L and by other immune proteins. It has been long known that RNase L is activated during the IFN response and that it can inhibit viral and bacterial infections, and cell proliferation. However, mapping the cellular mRNA targets that RNase L cleaves to achieve these effects proved challenging. We used a combination of RNA-seq strategies to identify the mRNA targets of RNase L and map its cellular program.
We found that RNase L cleaves the same mRNAs as are targeted by the microRNA miR-200. MiR-200 is a remarkable microRNA because it is a master regulator of cell adhesion, proliferation and is one of the key modulators of epithelial-to-mesenchymal transition (EMT) during metastasis. Thus, RNase L is functionally a mimic of miR-200. RNase L is unique because unlike miR-200, it is activated downstream of IFN and dsRNA signaling. RNase L could be approximately described as an "on-demand miR-200 in the innate immune system".
As expected from our analogy, RNase L and miR-200 have many similar effects on human cells. Both block proliferation, both block adhesion and both target ZEB1 transcription factor and regulate cadherins.
KO = knockout
RNA-SEQ OF RNASE L TARGETS IN T47D HUMAN BREAST CANCER CELLS
FDR = false discovery rate (<0.25% is significant; <0.05% is excellent)
RNase L cleaves common mRNA targets of select microRNAs and avoids ribosomal and mitochondrial protein mRNAs
GSEA and Rath-Korennykh gene signatures
TargetScan 6 gene signatures
Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome.
High-throughput RNA sequencing
Mammalian tissue culture
Our work is funded by Princeton University, Sidney Kimmel Foundation, Burroughs Wellcome Foundation, and NIH.