Tag Archives: molecular biology

It slices, it dices, and it protects the body from harm (Science)

By Catherine Zandonella, Office of the Dean for Research

RNase L enzyme structure

Researchers at Princeton have deciphered the 3D structure of RNase L, an enzyme that slices through RNA and is a first responder in the innate immune system. The structure contains two subunits, represented in red as two parts of a pair of scissors. Illustration by Sneha Rath, Inset courtesy of Science.

An essential weapon in the body’s fight against infection has come into sharper view. Researchers at Princeton University have discovered the 3D structure of an enzyme that cuts to ribbons the genetic material of viruses and helps defend against bacteria.

The discovery of the structure of this enzyme, a first-responder in the body’s “innate immune system,” could enable new strategies for fighting infectious agents and possibly prostate cancer and obesity. The work was published Feb. 27 in the journal Science.

Until now, the research community has lacked a structural model of the human form of this enzyme, known as RNase L, said Alexei Korennykh, an assistant professor of molecular biology and leader of the team that made the discovery.

“Now that we have the human RNase L structure, we can begin to understand the effects of carcinogenic mutations in the RNase L gene. For example, families with hereditary prostate cancers often carry genetic mutations in the region, or locus, encoding RNase L,” Korennykh said. The connection is so strong that the RNase L locus also goes by the name “hereditary prostate cancer 1.” The newly found structure reveals the positions of these mutations and explains why some of these mutations could be detrimental, perhaps leading to cancer, Korennykh said. RNase L is also essential for insulin function and has been implicated in obesity.

The Princeton team’s work has also led to new insights on the enzyme’s function.

The enzyme is an important player in the innate immune system, a rapid and broad response to invaders that includes the production of a molecule called interferon. Interferon relays distress signals from infected cells to neighboring healthy cells, thereby activating RNase L to turn on its ability to slice through RNA, a type of genetic material that is similar to DNA. The result is new cells armed for destruction of the foreign RNA.

The 3D structure uncovered by Korennykh and his team consists of two nearly identical subunits called protomers. The researchers found that one protomer finds and attaches to the RNA, while the other protomer snips it.

The initial protomer latches onto one of the four “letters” that make up the RNA code, in particular, the “U,” which stands for a component of RNA called uridine. The other protomer “counts” RNA letters starting from the U, skips exactly one letter, then cuts the RNA.

Although the enzyme can slice any RNA, even that of the body’s own cells, it only does so when activated by interferon.

“We were surprised to find that the two protomers were identical but have different roles, one binding and one slicing,” Korennykh said. “Enzymes usually have distinct sites that bind the substrate and catalyze reactions. In the case of RNase L, it appears that the same exact protein surface can do both binding and catalysis. One RNase L subunit randomly adopts a binding role, whereas the other identical subunit has no other choice but to do catalysis.”

To discover the enzyme’s structure, the researchers first created a crystal of the RNase L enzyme. The main challenge was finding the right combination of chemical treatments that would force the enzyme to crystallize without destroying it.

Korennykh groupAfter much trial and error and with the help of an automated system, postdoctoral research associate Jesse Donovan and graduate student Yuchen Han succeeded in making the crystals.

Next, the crystals were bombarded with powerful X-rays, which diffract when they hit the atoms in the crystal and form patterns indicative of the crystal’s structure. The diffraction patterns revealed how the atoms of RNase L are arranged in 3D space.

At the same time Sneha Rath, a graduate student in Korennykh’s laboratory, worked on understanding the RNA cleavage mechanism of RNase L using synthetic RNA fragments. Rath’s results matched the structural findings of Han and Donovan, and the two pieces of data ultimately revealed how RNase L cleaves its RNA targets.

Han, Donovan and Rath contributed equally to the paper and are listed as co-first authors.

Finally, senior research specialist Gena Whitney and graduate student Alisha Chitrakar conducted additional studies of RNase L in human cells, confirming the 3D structure.

Now that the human structure has been solved, researchers can explore ways to either enhance or dampen RNase L activity for medical and therapeutic uses, Korennykh said.

“This work illustrates the wonderful usefulness of doing both crystallography and careful kinetic and enzymatic studies at the same time,” said Peter Walter, professor of biochemistry and biophysics at the University of California-San Francisco School of Medicine. “Crystallography gives a static picture which becomes vastly enhanced by studies of the kinetics.”

Support for the work was provided by Princeton University.

Read the abstract.

Han, Yuchen, Jesse Donovan, Sneha Rath, Gena Whitney, Alisha Chitrakar, and Alexei Korennykh. Structure of Human RNase L Reveals the Basis for Regulated RNA Decay in the IFN Response Science 1249845. Published online 27 February 2014 [DOI:10.1126/science.1249845]

Shingles symptoms may be caused by neuronal short circuit (Proceedings of the National Academy of Sciences)

By Catherine Zandonella, Office of the Dean for Research

Neurons firing in synchrony could be responsible for pain, itch in shingles and herpes infection. Click to view movie. (Source: PNAS)

The pain and itching associated with shingles and herpes may be due to the virus causing a “short circuit” in the nerve cells that reach the skin, Princeton researchers have found.

This short circuit appears to cause repetitive, synchronized firing of nerve cells, the researchers reported in the journal Proceedings of the National Academy of Sciences. This cyclical firing may be the cause of the persistent itching and pain that are symptoms of oral and genital herpes as well as shingles and chicken pox, according to the researchers.

These diseases are all caused by viruses of the herpes family. Understanding how these viruses cause discomfort could lead to better strategies for treating symptoms.

The team studied what happens when a herpes virus infects neurons. For research purposes the investigators used a member of the herpes family called pseudorabies virus. Previous research indicated that these viruses can drill tiny holes in neurons, which pass messages in the form of electrical signals along long conduits known as axons.

The researchers’ findings indicate that electrical current can leak through these holes, or fusion pores, and spread to nearby neurons that were similarly damaged, causing the neurons to fire all at once rather than as needed. The pores were likely created for the purpose of infecting new cells, the researchers said.

Researchers at Princeton University imaged the synchronized, repetitive firing of herpes-infected neurons in a region known as the submandibular ganglia (SMG) between the salivary glands and the brain in mice. Image source: PNAS.

Researchers at Princeton University imaged the synchronized, repetitive firing of herpes-infected neurons in a region known as the submandibular ganglia (SMG) between the salivary glands and the brain in mice. (Source: PNAS)

The investigators observed the cyclical firing of neurons in a region called the submandibular ganglia between the salivary glands and the brain in mice using a technique called 2-photon microscopy and dyes that flash brightly when neurons fire. (Movie of synchronized firing of herpes-infected neurons.)

The team found that two viral proteins appear to work together to cause the simultaneous firing, according to Andréa Granstedt, who received her Ph.D. in molecular biology at Princeton in 2013 and is the first author on the article.  The team was led by Lynn Enquist, Princeton’s Henry L. Hillman Professor in Molecular Biology and a member of the Princeton Neuroscience Institute.

Each colored line and number on the right represents an individual neuron. The overlapping peaks indicate synchronized firing of neurons, which occurs when electrical current is able to leak from one neuron to the next. (Source: PNAS)

The first of these two proteins is called glycoprotein B, a fusion protein that drills the holes in the axon wall. A second protein, called Us9, acts as a shuttle that sends glycoprotein B into axons, according to the researchers. “The localization of glycoprotein B is crucial,” Granstedt said. “If glycoprotein B is present but not in the axons, the synchronized flashing won’t happen.”

The researchers succeeded in stopping the short circuit from occurring in engineered viruses that lacked the gene for either glycoprotein B or Us9. Such genetically altered viruses are important as research tools, Enquist said.

Finding a way to block the activity of the proteins could be a useful strategy for treating the pain and itching associated with herpes viral diseases, Enquist said. “If you could block fusion pore formation, you could stop the generation of the signal that is causing pain and discomfort,” he said.

Granstedt conducted the experiments with Jens-Bernhard Bosse, a postdoctoral research associate in molecular biology. Assistance with 2-photon microscopy was provided by Stephan Thiberge, director of the Bezos Center for Neural Circuit Dynamics at the Princeton Neuroscience Institute.

The team previously observed the synchronized firing in laboratory-grown neurons (PLoS Pathogens, 2009), but the new study expands on the previous work by observing the process in live mice and including the contribution of Us9, Granstedt said.

Shingles, which is caused by the virus herpes zoster and results in a painful rash, will afflict almost one out of three people in the United States over their lifetime. Genital herpes, which is caused by herpes simplex virus-2, affects about one out of six people ages 14 to 49 years in the United States, according the Centers for Disease Control and Prevention.

This research was funded by National Institutes of Health (NIH) Grants NS033506 and NS060699. The Imaging Core Facility at the Lewis-Sigler Institute is funded by NIH National Institute of General Medical Sciences Center Grant PM50 GM071508.

Read the abstract

Granstedt, Andréa E., Jens B. Bosse, Stephan Y. Thiberge, and Lynn W. Enquist. 2013. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins. PNAS 2013 ; published ahead of print August 26, 2013, doi:10.1073/pnas.1311062110

DNA Gridlock – Cells undo glitches to prevent mutations (Nature)

By Catherine Zandonella, Office of the Dean for Research

G4 Quadruplex

The diagram shows a G-quadruplex (G4) on the upper of the two strands that make up DNA. The purple shape represents DNA polymerase, which is blocked by the G4 in its attempt to copy DNA. Regions of the genome that are especially susceptible to forming G-quadruplexes are ones rich in guanine, which is one of the four nucleotides, designated by the letters G, A, C, and T, in DNA. Adapted from Nature Genetics, 2012.

Roughly six feet of DNA are packed into every human cell, so it is not surprising that our genetic material occasionally folds into odd shapes such as hairpins, crosses and clover leafs. But these structures can block the copying of DNA during cell division, leading to gene mutations that could have implications in cancer and aging.

Now researchers based at Princeton University have uncovered evidence that cells contain a built-in system for eliminating one of the worst of these roadblocks, a structure known as a G-quadruplex. In a paper published earlier this month in Nature, a group of researchers led by Princeton’s Virginia Zakian reported that an enzyme known as the Pif1 helicase can unfold these structures both in test tubes and in cells, bringing DNA replication back on track.

Given that Pif1 mutations have been associated with an increased risk of breast cancer, Zakian said, the study of how Pif1 ensures proper DNA replication could be relevant to human health. Zakian is Princeton’s Harry C. Wiess Professor in the Life Sciences.

Most DNA is made of up of two strands twisted about each other in a way that resembles a spiral staircase. Every time a cell divides, each DNA molecule must be duplicated, a process that involves unwinding the staircase so that an enzyme known as DNA polymerase can work down each strand, copying each letter in the DNA code. During this exposed period, regions of the unwound single strands can fold into G-quadruplexes (see diagram).

Like a car that encounters a pile-up on an Interstate, the DNA polymerase halts when it encounters a G-quadruplex, explained Matthew Bochman, a postdoctoral researcher who was a co-first author with Katrin Paeschke, now an independent investigator at University of Würzburg in Germany. The work also included Princeton graduate student Daniela Garcia.

“The DNA that is folded into a G-quadruplex cannot be replicated, so essentially it is skipped,” Bochman said. “Failure to copy specific areas of DNA that you really need is a serious problem, especially in regions that control genes that either suppress or contribute to cancer,” Bochman said.

Last year, the Zakian group in collaboration with human geneticists at the University of Washington reported that a mutation in human Pif1 is associated with an increased risk of breast cancer, suggesting that the ability to unwind G-quadruplexes could be important for protecting against cancer. The finding was published in the journal PLoS One.

G-quadruplexes could also be implicated in the process of aging, according to the researchers. The structures are thought to form at the ends of chromosomes in regions called telomeres, said Zakian, an expert on telomere biology.  Damaged or shortened telomeres are associated with premature aging and cancer.

To explore the role of Pif1 helicases in tackling G-quadruplexes, Bochman and Paeschke purified Pif1 helicases from yeast and bacteria and found that in test tubes, all of the Pif1 helicases unwind G-quadruplex structures extremely fast and very efficiently, much better than other helicases tested in the same way.

Next, these investigators set up an experiment to determine if Pif1 acts on G-quadruplexes inside cells. Using a system that could precisely evaluate the effects of G-quadruplex structures on the integrity of chromosomes, the researchers found that normal cells had no problem with the addition of a G-quadruplex structure, but when cells lack Pif1 helicases, the G-quadruplex induced a high amount of genome instability.

“To me, the most remarkable aspect of the study was the demonstration that Pif1-like helicases taken from species ranging from bacteria to humans and placed in yeast cells can suppress G-quadruplex-induced DNA damage,” Zakian said. “This finding suggests that resolving G-quadruplexes is an evolutionarily conserved function of Pif1 helicases.”

The Zakian lab also found that replicating through G-quadruplexes in the absence of Pif1 helicases results not only in mutations of the DNA at the site of the G-quadruplex but also in intriguing “epigenetic” effects on expression of nearby genes that were totally unexpected. Epigenetic events cause changes in gene expression that are inherited, yet they do not involve loss or mutation of DNA. Graduate student Daniela Garcia has proposed that the epigenetic silencing of gene expression that occurs near G-quadruplexes in the absence of Pif1 helicases is a result of the addition or removal of molecular tags on histones, which are proteins that bind DNA and regulate gene expression. This hypothesis is currently being studied.

The study involved contributions from Petr Cejka and Stephen C. Kowalczykowski of the University of California-Davis, and Katherine Friedman of Vanderbilt University.

Read the abstract.

Paeschke, Katrin, Matthew L. Bochman, P. Daniela Garcia, Petr Cejka, Katherine L. Friedman, Stephen C. Kowalczykowski & Virginia A. Zakian. Pif1 family helicases suppress genome instability at G-quadruplex motifs. Nature. 2013. doi:10.1038/nature12149.

This research was supported by the National Institutes of Health (V.A.Z., GM026938-34; S.C.K.GM041347), the National Science Foundation (K.L.F., MCB-0721595), the German Research Foundation (DFG), the New Jersey Commission on Cancer Research (K.P.) and the American Cancer Society (M.L.B., PF-10-145-02-01).

 

Herpes viruses commandeer protein production to storm neurons (Cell Host and Microbe)

By Morgan Kelly, Office of Communications

Viruses in the herpes family most commonly found in humans infect nervous system cells by “turning on” and then seizing control of the internal system these cells rely on to sense injury, among other signaling functions.

Princeton University researchers report in the journal Cell Host and Microbe that the pseudorabies virus (PRV) — a model herpes virus that infects animals — initiates and commandeers protein production in axons, the long offshoots of the cell body that connect neurons to other neurons and to tissue. After entering the neuron at the axon, the virus particles — which deliver the viral DNA that infects host cells — use the newly made proteins to travel to and infect the cell nucleus. Once there, the infection can spread to other neurons.

The research is the latest from the laboratory of senior researcher Lynn Enquist, the Henry L. Hillman Professor in Molecular Biology, to unravel the puzzling efficiency with which PRV and related herpes viruses invade the nervous system. PRV is an alpha-herpes virus, a prolific herpes subfamily that includes herpes simplex virus 1 (HSV-1), an extremely common human virus that causes cold sores and other lesions.

In the current paper, the researchers write that PRV “cleverly exploited” a natural cell process to speed up infection, a theme that resonates in past work from the Enquist lab on alpha-herpes viruses. In 2012, another researcher in the lab reported in Cell Host and Microbe that PRV and HSV-1 infections affect movement of neuronal mitochondria, the mobile organelles that regulate a cell’s energy supply, communication, and self-destruction response to infection.

For this newest research, Enquist worked with lead author Orkide Koyuncu, a postdoctoral research associate in molecular biology, and David Perlman, head of the molecular biology department’s mass spectrometry facility. They suggest that PRV particles first replicate in non-neuronal (such as skin and other tissue) cells at the site of body entry. The particles then enter axon terminals as the axon carries out its regular status-reports with those cells. The process of viral-particle entry is sensed by the neuron as a damage signal, which begins the protein production that will carry the virus particles to the nucleus.

Interestingly, the researchers discovered that the movement of incoming virus particles was disrupted by a genuine damage signal initiated before PRV infection. They hypothesized that the immediate response spurred by injury, infection or inflammation slows down other processes within the axon, which the researchers call “competitive inhibition.” When the molecular details of this crosstalk are fully understood, these signals could be used clinically to prevent the spread of alpha-herpes viruses.

Read the paper.

Citation: Koyuncu, Orkide O., David H. Perlman, Lynn W. Enquist. 2013. Efficient Retrograde Transport of Pseudorabies Virus within Neurons Requires Local Protein Synthesis in Axons. Cell Host & Microbe Vol. 13, no. 1, pp. 54-66.

This work was supported by U.S. National Institutes of Health grant R01NS033506-18.

A global post-transcriptional map of the elements that modulate RNA behavior (Nature)

Read the abstract here:
Systematic discovery of structural elements governing stability of mammalian messenger RNAs.
Goodarzi H, Najafabadi HS, Oikonomou P, Greco TM, Fish L, Salavati R, Cristea IM, Tavazoie S. Nature. 2012 Apr 8. doi: 10.1038/nature11013. [Epub ahead of print]