Tom Cech

Tom Cech publishes new book, The Catalyst: RNA and the Quest to Unlock Life's Deepest Secrets!

Anonymous — Mon, 03 Jun 2024 13:10:54 +0000

Tom Cech publishes new book, The Catalyst: RNA and the Quest to Unlock Life's Deepest Secrets! Anonymous (not verified) Mon, 06/03/2024 - 07:10

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How does a stem cell know what to become? Study shows RNA plays key role

Anonymous — Tue, 07 Jul 2020 18:24:26 +0000

How does a stem cell know what to become? Study shows RNA plays key role Anonymous (not verified) Tue, 07/07/2020 - 12:24

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C9orf72 and triplet repeat disorder RNAs: G-quadruplex formation, binding to PRC2 and implications for disease mechanisms.

Anonymous — Tue, 22 Oct 2019 17:45:49 +0000

C9orf72 and triplet repeat disorder RNAs: G-quadruplex formation, binding to PRC2 and implications for disease mechanisms. Anonymous (not verified) Tue, 10/22/2019 - 11:45

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNA-binding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G₄C₂)_n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.

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Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines

Anonymous — Tue, 22 Oct 2019 17:43:14 +0000

Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines Anonymous (not verified) Tue, 10/22/2019 - 11:43

Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. In comparison, little is known about TERT expression at the single-cell and single-molecule level. To address this, we analyzed TERT expression across 10 human cancer lines using single-molecule RNA fluorescent in situ hybridization (FISH) and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, spliced TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that spliced mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells.

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Regulation of histone methylation by automethylation of PRC2

Anonymous — Tue, 22 Oct 2019 17:41:16 +0000

Regulation of histone methylation by automethylation of PRC2 Anonymous (not verified) Tue, 10/22/2019 - 11:41

Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that is critical for regulating transcriptional repression in mammals. Its catalytic subunit, EZH2, is responsible for the trimethylation of H3K27 and also undergoes automethylation. Using mass spectrometry analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, and K515) on a disordered but highly conserved loop of EZH2. Methylation of these lysines increases PRC2 histone methyltransferase activity, whereas their mutation decreases activity in vitro. De novo histone methylation in an EZH2 knockout cell line is greatly impeded by mutation of the automethylation lysines. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on a flexible loop. This posttranslational modification and cis regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We propose that EZH2 automethylation allows PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.

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The unexpected complexities of TERT, a key cancer driver

Anonymous — Wed, 11 Sep 2019 06:00:00 +0000

The unexpected complexities of TERT, a key cancer driver Anonymous (not verified) Wed, 09/11/2019 - 00:00

Cu Boulder Today

Telomerase reverse transcriptase (TERT), an enzyme associated with nearly all malignant human cancers, is even more diverse and unconventional than previously realized, new University of Colorado Boulder research finds.

Telomeres, the protective ends of chromosomes, help to maintain genomic stability. In most normal adult human cells, the telomeres eventually shorten beyond a critical length, bringing a cell’s life to its natural end. In almost all human cancers, however, telomerase reactivates, leading to cell proliferation and tumor formation. This is a key part of what makes cancer cells “immortal.”

As TERT is the component of telomerase that is required for cancer development, it has become an attractive target for cancer therapeutics in recent decades.

“TERT has great importance in cancer progression and hence there is a great interest in studying its expression regulation,” said Gabrijela Dumbovic, a post-doctoral researcher in the Department of Biochemistry and co-lead author of the CU Boulder study.

The study, published recently in the journal Proceedings of the National Academy of Sciences, used powerful, high-magnification imaging techniques to reveal differences in how TERT is produced on the single-cell level, in individual cancer cells. Previously, studies relied on general averages of TERT production within whole tissue samples or large cell populations, without investigating potential cell-to-cell variation.

About a year ago, researchers at the BioFrontiers Institute began discussing ways to apply ribonucleic acid (RNA) localization imaging protocols to better classify how TERT RNA was being made within individual cancer cells. (RNA typically serves as a messenger for turning DNA genetic information into proteins, though it is also important for coding and regulating gene expression and catalyzing certain biochemical reactions.) What began as a casual conversation grew into an interdisciplinary effort, combining experience in RNA imaging from the laboratory of Professor John Rinn of CU Boulder’s Department of Biochemistry and the BioFrontiers Institute with innovative telomerase research led by Distinguished Professor Thomas Cech, a Howard Hughes Medical Institute (HHMI) Investigator and Nobel laureate.

“We have a great community of scholars in the Caruthers Biotechnology Building,” Cech explained. “Our students and postdoctoral fellows are encouraged to talk freely about their work, so collaborations are forged naturally and frequently.”

The researchers used a microscopy technique known as single-molecule RNA fluorescent in situ hybridization (“smFISH,” for short) to visualize individual RNA molecules that coded for TERT in separate cancer cells. Looking at the microscope images, they counted the number of TERT RNA molecules – which appeared as tiny fluorescent spots – in different locations within each cell.

“Previous studies were mostly focused on studying TERT expression in a population of cells. We took a different approach and actually could visualize TERT RNA levels, and RNA distribution on a single-cell level,” said Dumbovic.

The authors also found that while most cells in our bodies have two copies of a given gene (one from our mother and one from our father), the cancer cells frequently had more than two copies of the TERT gene. Such gene amplification is common in cancer cells, which have relatively unstable genomes.

“The TERT gene has made all these extra copies of itself,” Rinn said. “Selfishly, it wants to replicate itself, and cancer wants to hijack that mechanism to keep the its cells alive indefinitely. That’s something we can only see with this kind of imaging.”

Intriguingly, the study also found that when looking at where TERT messenger RNA resides within a given cell, a high amount (over 80 percent in some instances) stays quarantined in the nucleus, rather than the expected cytoplasm, raising yet another mystery for future study. Typically, messenger RNA, which is made in the cell’s nucleus, is exported from the nucleus to be turned into protein in the cell’s cytoplasm.

“RNA imaging has continually shed new insights into biology by providing an important layer of information of where a gene is in the cell. When we took the ‘molecular picture’ of the TERT gene, we’re struck by the unexpected and sometimes substantial amount of TERT RNA in the nucleus where it can’t function to make protein,” explained Rinn. “This opens up a new layer of regulation where the molecules of TERT RNA are when considering its abundance in cancer and other disease states.”

This surprising pattern of nuclear localization was also observed in healthy cells that produce TERT, specifically human induced pluripotent stem cells (iPSCs). “This suggests that the nuclear localization is not a behavior specific to the diseased cancer cells,” said Teisha Rowland, co-lead author of the study and former post-doctoral research in the lab of Thomas Cech. Rowland is now Director of the new Stem Cell Research and Technology Resource Center in the Department of Molecular, Cellular, and Developmental Biology.

“It was generally assumed that TERT mRNA localizes to the cytoplasm, which is needed for it to make a protein product, but now we have a different perspective. Now, we want to understand why TERT RNA is retained in the nucleus,” Dumbovic said. “Is there a stimulus that causes it to move, or to stay?”

The research adds to the increasingly complex picture of TERT’s role in making cancer cells immortal, nuances that could lead to more effective therapeutic solutions for cancer in the future.

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Nobel Laureate, Tom Cech, Ph.D., suggests new way to target third most common oncogene, TERT

Anonymous — Tue, 10 Sep 2019 06:00:00 +0000

Nobel Laureate, Tom Cech, Ph.D., suggests new way to target third most common oncogene, TERT Anonymous (not verified) Tue, 09/10/2019 - 00:00

Garth Sundem

Healthy cells have a built-in self-destruct mechanism: Strands of DNA called "telomeres" act as protective caps on the ends of your chromosomes. Each time a cell replicates, telomeres get a little shorter. Think of it like filing your nails with an Emory board - after enough filing, you hit your fingertip - ouch! In the case of healthy cells, after enough replications, telomeres are "filed" away, leaving bare ends of the chromosomes exposed. At that point, healthy cells are inactivated or die. The eventual loss of telomeres is a major reason you are not immortal. This cellular mortality is also a major way your body fights cancer.

That's because a hallmark of cancer is cellular immortality. And for that to happen, somehow, some way, cancer cells have to break the body's system of telomere degradation - cancer needs to keep chromosomes safely capped. One way they do it is by spackling new DNA onto telomeres faster than it's lost. This involves supercharging the gene that codes for new telomere material.

The TERT gene is the third most commonly mutated gene in cancer. When cancer over-activates TERT, it manufactures more of the enzyme "telomerase," which rebuilds telomeres faster than they are degraded. With telomeres being built faster than they degrade, cancer cells gain immortality. Especially cancer like melanoma, glioblastoma, and bladder cancers (among others) are defined by TERT mutation. It's likely that without TERT mutation, there would be none of these cancers.

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A key ‘kill switch’ in a gene-regulating protein group

Anonymous — Mon, 09 Sep 2019 06:00:00 +0000

A key ‘kill switch’ in a gene-regulating protein group Anonymous (not verified) Mon, 09/09/2019 - 00:00

Trent Knoss

CU Boulder and Howard Hughes Medical Institute (HHMI) biochemists have revealed a key regulatory process in a gene-suppressing protein group that could hold future applications for drug discovery and clinical treatment of diseases, including cancer.

The new research, recently published in the journal Genes & Development, centered on a protein group known as Polycomb Repressive Complex 2 (PRC2), which acts as a gatekeeper for gene expression as cells differentiate and tissues develop.

“PRC2 plays a critical role in stem cell differentiation to make sure that irrelevant genes are switched off,” said Yicheng Long, an HHMI post-doctoral fellow and a co-author of the study. “If you have a muscle cell, for example, PRC2 shuts off genes that are specific to the brain.”

When that regulation goes awry, however, abnormal PRC2 activation is suspected to play a role in the development of diseases such as cardiac hypertrophy, Huntington’s Disease and multiple types of cancer.

Researchers from HHMI and CU Boulder’s Department of Biochemistry began by re-examining exactly how PRC2 achieves methylation, a complex epigenetic process by which proteins modify the structure of regions of chromosomes.

While examining the activity of human PRC2, the scientists began to notice a “mystery band” appearing in the data. As PRC2 was previously known to modify an important histone protein that is a fundamental unit of human chromosome, the scientists indeed observed this modification in vitro. Surprisingly, the scientists noticed another modification event indicated by this “mystery band.” Although other scientists had seen this band before, nobody could understand how and why it was happening.

“This unexpected band caught our attention and we suspect that this could represent a novel activity and function of PRC2,” said Xueyin Wang, one of the two co-first authors of the study and a then-CU Boulder graduate student now with A2 Biotherapeutics Inc. in California.

Further investigation revealed that this “mystery band” is a self-modification event (named “automethylation”) which have important physiological functions. Using mass spectrometry, it became apparent that PRC2 automethylates three lysines of a flexible, evolutionarily conserved loop. The loop essentially holds the key to its own lock within its own structure and remains poised in an inhibited state. Automethylation of the three lysines unlocks this loop from PRC2’s catalytic center and thus relieves PRC2 from the poised state.

“The interesting question is why nature would devise such a mechanism,” Long said.

The researchers hypothesize that with abundant level of PRC2 in stem cell, the flexible loop ensures that most of it stays inactive until needed, like a fire sprinkler that stays closed during normal operations, only opening when a fire needs to be extinguished. If that sprinkler ever malfunctions and remains open (as in a cancerous mutation), biochemists can now foresee a means of re-closing it to prevent unwanted flooding.

“Others have found the way to activate PRC2,” Long said. “We found a key to turning it off.”

“I expect that many other examples of automethylation will be found,” said Nobel Laureate and Distinguished Professor Thomas Cech, the senior author of the study and an HHMI Investigator. “Many enzymes that regulate our genes do so by adding methyl groups to their target proteins. So they’re also primed to add methyl groups to themselves, allowing them to self-regulate their own activity.”

With greater knowledge of PRC2’s form and function, the research could one day lead to more specific clinical focus on inhibiting activations associated with tumor formation and other known disease pathways.

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Faculty in Focus: Tom Cech looks back on Nobel Prize in chemistry

Anonymous — Tue, 01 Jan 2019 06:00:00 +0000

Faculty in Focus: Tom Cech looks back on Nobel Prize in chemistry Anonymous (not verified) Mon, 12/31/2018 - 23:00

Kenna Bruner

It’s been 30 years since CU Boulder Distinguished Professor Tom Cech received the 1989 Nobel Prize in chemistry for his findings that RNA in living cells is not only a molecule that encodes information but can also function as a catalyst. His discovery laid the foundation for advances in molecular genetics and gave rise to an expanding appreciation of the roles of RNA in biology.

Among his many awards and recognitions, Cech was selected as a Howard Hughes Medical Institute (HHMI) investigator in 1988 and served as president of the Chevy Chase, Maryland-based HHMI—the nation’s largest private supporter of basic biomedical research—from 2000 to 2009, while retaining his CU Boulder faculty positions and lab. He is an HHMI investigator and the director of the CU Boulder BioFrontiers Institute and also has a faculty appointment at the CU Anschutz Medical Campus.

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Video: Nobel laureate Tom Cech still loves teaching

Anonymous — Mon, 15 Oct 2018 06:00:00 +0000

Video: Nobel laureate Tom Cech still loves teaching Anonymous (not verified) Mon, 10/15/2018 - 00:00

CU Boulder's Tom Cech won the Nobel Prize for Chemistry in 1989, but he firmly believes his place is still in the classroom teaching undergraduates. Here, he discusses how teaching adds meaning to his life and how he still works to become a better teacher.

[video:https://www.youtube.com/watch?v=k3yqYgjNBgk]

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