Visualizing and quantifying frameshifting mechanisms in living cells

Many infectious viruses, from HIV to West Nile, rely on a fundamental biological process called frameshifting to maximize their attack. Long identified as a key mechanism that viruses use to proliferate inside their hosts, the real-time dynamics of frameshifting had never been directly observed, until now.

For the first time, Colorado State University scientists Tim Stasevich and Brian Munsky have developed detailed imaging technology and computational analyses to visualize, quantify and understand frameshifting mechanisms at the level of single molecules in living cells.

The publication of their work June 6 in the journal Molecular Cell includes the first author and graduate student Kenneth Lyon, and computational modeling support from postdoctoral researcher Luis Aguilera. The research is supported by a $1.2 million grant from the W. M. Keck Foundation.  {module In-article}

"Our hope is that these integrated experimental and computational methods, allowing us to observe and predict unique aspects of viral replication, can offer strategies for future antiviral therapies," said Stasevich, a Monfort Professor and assistant professor in the Department of Biochemistry and Molecular Biology. 

HIV is one example of a retrovirus, meaning it carries genetic information in a molecule called RNA, rather than DNA. When a virus infects a host cell, it makes viral proteins by the manipulation of ribosomes, the cellular protein synthesis machines in which genetic instructions are converted, or translated, from RNA into proteins. During frameshifting, a ribosome that's translating an RNA "slips" one spot backward or forward along a nucleotide sequence, resulting in the translation of an entirely different protein sequence moving forward. This process essentially nets two proteins for the price of one RNA, and it allows viruses to keep their genomes efficiently compact.

Stasevich has been a pioneer in molecular imaging, using engineered protein tags that selectively bind and fluoresce in different colors when, for example, RNA translation occurs. His lab has developed sensitive microscopes that capture these binding events in real time, making movies of heretofore invisible processes. Munsky, a former Richard P. Feynman Fellow at Los Alamos National Laboratory and assistant professor in the Department of Chemical and Biological Engineering, is an expert in building computational models to sift through noisy or "stochastic" single-molecule data to find statistical signatures of hidden biophysical mechanisms

In this latest experiment, Stasevich's team simultaneously monitored the translation of single RNAs into two, unique protein chains during frameshifting, using the HIV-1 virus's binding fragments (not the whole virus).

Peering into this before-hidden world, what the team saw might surprise some biologists.

They found that frameshifting occurs in bursts of activity, after a longer period of non-frameshifting.

It was previously known that, for instance, the HIV virus outputs frameshifted proteins about 5 percent of the time. But instead of lots of RNAs frameshifting at once, the researchers observed a subset of RNAs frameshifting like mad, with about 5 percent of the RNA doing all the frameshifting, as opposed to 100 percent of the RNA sharing frameshifting duties. This demonstrates that frameshifting happens in only a small subset of RNA.

It remains unclear what exactly distinguishes this frameshifting RNA from others, the researchers say. But their newfound ability to target this special subset of viral RNA promises new depth to the understanding of viral replication and could someday inform new antiviral therapeutics.

Munsky's modeling group recreated all the Stasevich lab's frameshifting observations through detailed computer simulations of ribosome traffic along with RNA molecules. Aguilera and Munsky showed that temporal fluctuations in the fluorescent data could be reproduced only if ribosomes involved in viral frameshifting sometimes pause at the frameshift site. Their models suggested that these pauses would induce ribosomal "traffic jams" that maintain production of frameshifted proteins long after cessation of regular translation. The models predicted, and imaging experiments validated, that moving the frameshift site to the middle of the RNA induced bigger "traffic jams."

The researchers' next goal is to simultaneously visualize many more - perhaps hundreds - of different RNA and protein molecules, each with their own fingerprint of color, brightness pattern or fluctuation speed. These and other statistical signatures can be visualized and quantified through the team's continued close integration of single-molecule imaging and computational modeling.

The researchers said they are grateful for the Keck Foundation's pivotal role in allowing Stasevich and Munsky's teams a unique opportunity to combine their complementary areas of expertise. "We see this as a long-term collaboration," Stasevich said.

University of Lincoln research looks at the possibility of moons outside our solar system causing gaps in the rings of planet J1407b

Moons orbiting planets outside our solar system could offer another clue about the pool of worlds that may be home to extra-terrestrial life, according to an astrophysicist at the University of Lincoln.

Exoplanets are planets outside our solar system and up to this point, nearly 4,000 have been discovered. Only a small proportion of these are likely to be able to sustain life, existing in what is known as the habitable zone. But some planets, especially large gas giants, may harbor moons which contain liquid water.

Dr. Sutton said: "These moons can be internally heated by the gravitational pull of the planet they orbit, which can lead to them having liquid water well outside the normal narrow habitable zone for planets that we are currently trying to find Earth-like planets in. I believe that if we can find them, moons offer a more promising avenue to finding extra-terrestrial life." {module In-article}

This interest has inspired Dr. Sutton's latest research, which looked at the possibility of moons orbiting the exoplanet J1407b, analyzing whether they may have caused gaps in the planet's ring system.

Because of their size and distance from Earth, exomoons are very difficult to detect. Scientists have to locate them by looking for the effect they have on objects around them, such as planetary rings.

Dr. Sutton ran supercomputer simulations to model the rings around J1407b, which are 200 times larger than those around Saturn. Gravitational forces between all particles were calculated and used to update the positions, velocities, and accelerations in the computer models of the planet and its ring system. He then added a moon that orbited at various ratios outside of the rings to test whether this caused gaps to form where expected over 100 orbital periods.

Findings revealed that while the orbiting moon did have an effect on the scattering of particles along the ring edge, the expected gaps in the ring structure were unlikely to be caused by the gravitational forces of a currently unseen moon orbiting outside the rings.