Noncoding RNA in biology, biotechnology and human disease
RNA is one of the most fascinating molecules in biology. It seems there is no job that RNA doesn't have a stake in, from transcription to translation, catalysis to structure, information storage to ligand binding -- designed by nature to be versatile and multipurpose. Having so many irons in the fire, however, means that RNA also has a part to play when processes go awry in disease. We are interested in the biology and biochemistry of ncRNA, particularly in the nucleus. Projects in the Gagnon lab surrounding this subject area are broadly divided into these three topics:
1. RNA-centric molecular disease mechanisms and therapeutics for C9FTD/ALS.
2. Structure-function studies and precision engineering of RNA-guided enzymes.
3. Genomics, transcriptomics, and epitranscriptomics of pathogenic RNA viruses and host interactions.
4. Noncoding RNA (ncRNA) on the chromatin and in nuclear organization.
1. RNA-centric molecular disease mechanisms and therapeutics for C9FTD/ALS.
2. Structure-function studies and precision engineering of RNA-guided enzymes.
3. Genomics, transcriptomics, and epitranscriptomics of pathogenic RNA viruses and host interactions.
4. Noncoding RNA (ncRNA) on the chromatin and in nuclear organization.
RNA-centric molecular disease mechanisms and therapeutics for C9FTD/ALS.
Our primary focus is a neurological repeat expansion disease known as C9FTD/ALS, the leading genetic cause of amyotrophic lateral sclerosis (ALS, or Lou Gehrig's Disease) and frontotemporal dementia (FTD). C9FTD/ALS is caused by the expansion of a (GGGGCC) hexanucleotide repeat in the first intron of a C9ORF72 transcript variant that is transcribed into expanded tandem repeat-containing RNA (xtrRNA). The repeat can be transcribed in both the sense and antisense (CCCCGG) direction. The expansions may range from under one hundred repeats to thousands of repeats. These xtrRNAs form foci in the nucleus of cells, may fold into unusual non-canonical RNA structures like G-quadruplexes, and can sequester RNA-binding proteins and functionally deplete them. These xtrRNAs can also be exported and translated in the cytoplasm, resulting in production of toxic poly-dipeptides that contribute to the molecular disease state. These seem to include defects in nucleocytoplasmic transport, autophagy, protein homeostasis, RNA homeostasis, dynamics of membrane-free organelles, cellular trafficking, cellular energetics, and RNA metabolism. We are focusing our efforts on three fronts:
i) Developing cell-based and in vitro models of disease to enable biochemical and mechanistic studies.
ii) Understanding how the cells deal with xtrRNA, including synthesis, transport, translation and decay.
iii) Rational design and unbiased screens to identify small molecules and biologics of therapeutic value.
Structure-function studies and precision engineering of RNA-guided enzymes.
We have a long-standing interest in the structure, function, evolution, and application of RNA-guided enzymes like Argonaute and the CRISPR-associated (Cas) proteins. In particular, we have been quite preoccupied with CRISPR, which has emerged as a leading biotechnology and potential therapeutic approach. We are currently investigating the structure-function relationships and conformational transitions in CRISPR-Cas9 and CRISPR-Cas12a. We are also exploring tools and therapeutic applications that can come out of chemical and genetic manipulations of CRISPR. To test our hypotheses, we participate in several collaborations to combine biochemistry with computational and synthetic chemistry, high-throughput screening, and molecular and directed evolution. We expect to uncover fundamental principles of RNA-guided enzymes that will enable creation of tailor-made enzymes to control nearly any pathway involving nucleic acids in biology and biotechnology. Experimental areas we are currently focusing on are:
i) Chemical modification of CRISPR RNA guides.
ii) Chemical and biologic inhibitors of CRISPR enzymes.
iii) Induced-fit mechanisms and RNP assembly of CRISPR.
iv) Cas enzyme engineering for improved or novel applications.
Genomics, transcriptomics, and epitranscriptomics of pathogenic RNA viruses.
When COVID-19 began, our laboratory decided to help fight the pandemic by sequencing the genome of the virus that causes it, SARS-CoV-2. Near real-time sequencing of samples from the state of Illinois helped track the introduction and spread of new variants. It also allowed us to identify and characterize a new variant, now called B.1.2 or 20G, that was dominant within and unique to the U.S. A key feature of our sequencing has been the use of long-read nanopore sequencing. We are continuing to expand our new-found interest in RNA viruses and nanopore sequencing by applying these approaches to the role of HIV-1 transcript modification and SARS-CoV-2 transcript processing.
Noncoding RNA (ncRNA) on the chromatin and in nuclear organization.
With the advent of modern sequencing technology, it has become apparent that thousands of ncRNAs may exist that play important, uncharacterized functions in human biology. Of particular interest are novel roles for long ncRNAs (lncRNAs) that may act on the chromatin. A common theme for ncRNA is serving in the roles of structural scaffolds to assemble higher-order ribonucleoprotein complexes, as well as guides to direct the activity and function of associated proteins. The evolution and conservation (or lack thereof) of these RNAs is of particular interest with respect to their emergence and their roles in human biology and disease. We seek to correlate lncRNA expression and biochemical function with basic and highly conserved cellular functions, such as cell division and neuronal differentiation. Two approaches we are taking are:
i) Global RNA localization on chromatin during cellular processes using methods like iMARGI.
ii) The role of ncRNA in nuclear structural organization and regulation.