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Printable Handouts
Navigable Slide Index
- Introduction
- Epitranscriptomic modification
- m6A addition to RNA transcripts
- Subcellular localization of the m6A writers and readers
- IAV infection can induce both m6A writers and readers
- Overexpression of YTHDF2 strongly enhances IAV replication
- Loss of the m6A methyltransferase
- Mapping m6A sites on IAV mRNAs by PAR-CLIP
- m6A sites on the IAV HA segment
- Loss of m6A sites on the HA segment reduces IAV pathogenicity
- Inhibition of RNA methylation
- 3-deazaadenosine (DAA)
- Infected A549 cells treated with DAA
- Conclusion: m6A and IAV
- Epitranscriptomic modifications in HIV-1
- UPLC-MS/MS analysis of purified HIV-1 gRNA
- Hypothesis 1
- Mapping m6A sites in the 3’ UTR of HIV-1
- Question
- Does YTHDF2 expression affect the rate of HIV-1 replication in CD4+ T-cells?
- YTHDF2 promotes HIV-1 replication in CD4+ T-cells
- The effect of DAA treatment
- Hypothesis 2
- YTHDF2 overexpression promotes SV40 replication
- Loss of YTHDF2 or METTL3 inhibits SV40 replication
- DAA and SV40 replication
- Conclusions: m6A modifications
- m5C may promote viral gene expression
- Identifying the HIV-1 RNA m5C writer
- NSUN2 is the major m5C writer for HIV-1 RNAs
- NSUN2 CLIP and knockout using CRISPR/Cas
- Conclusions: m5C and NSUN2
- Loss of NSUN2 inhibits HIV-1 replication
- NSUN2 mutants and HIV-1 gene expression
- m5C sites in infected T-cells
- NSUN2 knockout and ribosomal association
- Conclusions: m5C modifications
- N4-acetylcytidine (ac4C)
- ac4C modification sites
- NAT10 adds ac4C to HIV RNA transcripts
- ac4C & NAT10 binding sites
- Overexpression of WT but not mutant NAT10 increases HIV-1 gene expression
- HIV-1 single-round infection in ΔNAT10 CEM T-cells
- Viral RNA expression and NAT10
- Spreading HIV-1 infection in ΔNAT10 CEM T-cells at 3dpi
- Inhibition of NAT10 function
- Silent mutagenesis of ac4C sites in HIV-1 env inhibits Gag expression in cis
- Conclusions: ac4C modification
- Summary
Topics Covered
- Epitranscriptomic modifications (m6A, m5C and ac4c modifications)
- mRNA epitranscriptomic modification
- Loss of m6A modifiers and IVA replication
- m6A modifiers: YTHDF proteins and METTL3
- Epitranscriptomic modifications in HIV-1
- m5C modifiers: NSUN2
- ac4c modifiers: NAT10
- m6A and ac4c increase viral mRNA stability while m5C increases viral mRNA translation
Links
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Therapeutic Areas:
Talk Citation
Cullen, B.R. (2021, February 25). Viral epitranscriptomics [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved December 13, 2024, from https://doi.org/10.69645/SXQS1281.Export Citation (RIS)
Publication History
Financial Disclosures
- Professor Bryan R. Cullen has a patent application pending that claims the use of mRNA epitranscriptomic modifications, specifically m6A, as a way to boost mRNA expression in cis, by addition of m6A sites to the mRNA, and/or in trans, by overexpression of the m6A reader YTHDF2. He does not have any biotech involvement at present.
A selection of talks on Genetics & Epigenetics
Transcript
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0:00
Hello, my name is Bryan Cullen.
I'm a professor in the Department of Molecular Genetics and
Microbiology at Duke University Medical Center in Durham, North Carolina.
The title of my talk today is viral epitranscriptomics.
What is epitranscriptomics?
The term is derived from the term epigenetics,
and is similar in that it refers to information
in the case of epitranscriptomics on RNA and
in the case of epigenetics on the DNA, that's not hardwired into the sequence of a gene.
0:29
If we look at viral and cellular mRNAs,
it's known that they are subject to what is called epitranscriptomic modification.
There are several different covalent modifications,
primarily methylations but also acetylations, of individual nucleotides in
eukaryotic mRNAs that have been detected at levels
greater than 0.05 percent of the parental nucleotide.
Of these, the most common is addition of a methyl group
to the N6 position of adenosine called m_6A,
but methylation of the C5 position of cytidine m_5C,
acetylation of the N4 position of cytidine,
ac4C, and methylation of the ribose moiety for all four bases
(this is called 2'-O-methyl modification
collectively abbreviated as Nm) are also prevalent.
The presence of high levels of m_6A in viral mRNAs was first
reported for influenza virus by Krug et al in 1976,
but no further progress was made on the significance of these modifications.
However, the modification was subsequently
identified in a range of viruses including retroviruses,
and as I said, the function of m_6A if anything remained unclear.
1:39
This schematic shows what has been uncovered about
m_6A addition over the last 10 years or so, by a number of different groups.
We know that there is a complex of
at least three proteins that adds m_6A to mRNAs as they're transcribed,
so it's a co-transcriptional process.
These m_6A groups can be recognized in
the nucleus by two proteins referred to as readers,
called YTHDC1 and YTHDC2,
but the more important readers are in fact in the cytoplasm and interact
with m_6A sites on mRNA after nuclear export of that mRNA.
These cytoplasmic readers are referred to YTHDF1, YTHDF2 and YTHDF3.
The YTHD part of their names refers
to the unit within the protein that actually binds to m_6A,
and that has been crystallized,
so it's clear that there's a very direct interaction between m_6A and the YTHDF domain.