Regulatory non-coding RNA
The central aim of the Shkumatava laboratory is to understand the cellular and molecular functions of regulatory non-coding RNA. Regulatory non-coding RNA comes in many flavours from small to long RNAs and is also embedded in coding RNAs from messenger RNA to viral transcripts. These regulatory RNAs control diverse developmental, physiological and pathological processes across the kingdom of life.
lncRNAs
Long non-coding RNAs (lncRNAs) are non-coding RNAs transcribed by RNA Polymerase II with a length greater than 200 nucleotides and limited or no coding potential. They occupy a vast proportion of eukaryotic genome transcriptional units and regulate important cellular processes. However, the principles underlying their molecular functions are often enigmatic. We use lncRNAs as a paradigm to identify functional non-coding RNA elements and to discover their molecular mechanism of action.
Conserved sequence elements of lncRNAs
One strategy to identify functional non-coding RNA elements is through sequence conservation. However, lncRNA sequences undergo rapid evolutionary turnover resulting in only a small set of ‘ultraconserved’ lncRNAs that show detectable sequence conservation in vertebrates1. Using this approach, we discovered a conserved element within a lncRNA that regulates animal behaviour from zebrafish to mouse. The element functions through nearly perfect base-complementarity with a microRNA that mediates targeted destruction of the microRNA2. We continue our analysis of ultraconserved sequence elements to discover lncRNAs and embedded regulatory elements with key cellular functions.
Functional conservation of lncRNAs with divergent sequences
Synteny is another starting point to identify functionally important lncRNAs. Most syntologous lncRNAs separated by substantial evolutionary distances do not exhibit detectable sequence conservation. Nonetheless, complementation experiments showed that these divergent lncRNA sequences can carry out conserved biological functions, however the cryptic determinants of their function are not fully understood. In addition to guiding RNA binding, lncRNA can also function through interaction with proteins. We devised the incPRINT methodology which allows for the identification of RNA specific RNA-binding proteins3. Using this approach, we found novel interactors and effectors of Xist, the lncRNA essential for mammalian sex chromosome dosage compensation3. In addition to linear RNA sequence motifs that are recognised by some proteins, structural RNA elements are also the basis of RNA-protein and RNA-RNA interactions. We recently found that syntologous lncRNAs with no detectable sequence homology carry out similar biological functions mediated by a set of conserved lncRNA-protein interactions4. One of current aims is to define the cryptic determinants of non-sequence conserved non-coding regulatory RNA function.
Viral non-coding RNA
DNA and RNA viruses rely on regulatory non-coding RNA for their replication and host infection. Viral 5'- and 3'- untranslated regions (UTRs) contain specific regulatory sequence motifs whose recognition by proteins impacts post-transcriptional regulation of gene expression, modulation of subcellular localization, translation efficiency, and RNA stability. The identification of such sequence motifs and associated host RNA-binding proteins therefore both present an opportunity to understand essential viral-host mechanisms and discover potential vulnerabilities in viral infection, replication and immune suppression. We identified a set of human proteins associated within the untranslated regions of SARS-CoV-2 by employing quantitative incPRINT. This approach revealed host RNA-binding proteins supporting or inhibiting viral propagation. We currently strive to expand this approach to other viruses with similar host-switching potential. The identification of conserved regulatory noncoding RNA sequences, RNA structural elements and associated proteins is an important asset in understanding viral transmission and can enable preparation for future pandemics.
Selected publications:
1. Ulitsky, I., Shkumatava, A., Jan, C.H., Sive, H. & Bartel, D.P. Conserved function of lincRNAs in vertebrate embryonic development despite rapid sequence evolution. Cell 147, 1537-50 (2011).
2. Bitetti, A. et al. MicroRNA degradation by a conserved target RNA regulates animal behavior. Nat Struct Mol Biol 25, 244-251 (2018).
3. Graindorge, A. et al. In-cell identification and measurement of RNA-protein interactions. Nat Commun 10, 5317 (2019).
4. Sabate-Cadenas, X. et al. Conserved RNA-binding protein interactions mediate syntologous lncRNA functions. bioRxiv, 2024.08.21.605776 (2024).