KANAZAWA, Japan, July 29, 2024 /PRNewswire/ -- Researchers at Nano
Life Science Institute (WPI-NanoLSI), Kanazawa University, IMDEA
Nanoscience (Madrid, Spain) and
CNB-CSIC (Madrid, Spain) report in
ACS Nano experiments that reveal a cycle of
conformational stages that recombinant Influenza A genomes pass
through during RNA synthesis.
Influenza A is a global health risk responsible for local
epidemics and deadly pandemics. As such, the mechanism by which the
virus replicates itself has attracted significant interest.
Researchers led by Shingo Fukuda at
Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University in
Japan, Jaime Martin-Benito at CNB-CSIC and Borja Ibarra at IMDEA Nanociencia in
Spain, used high-speed atomic
force microscopy and electron microscopy to pin down the
conformational dynamics of recombinant viral genomes (or rRNPs)
during RNA synthesis.
Previous attempts to understand what possible conformational
changes occur during Influenza A viral multiplication cycle had
been hindered by what the researchers describe as the "bulky
double-helical structure" of the viral RNPs (vRNP, Fig.1 A),
which made it hard to see what was going on. As a result the
researchers produced a circular recombinant ribonucleoprotein
complex (rRNP) (Fig.1 B), which allowed them to overcome the
'bulky issue'. The authors used HS-AFM to follow conformational
changes of individual rRNP complexes in real-time during active RNA
synthesis (Fig.1 C).
Their work provides first direct experimental evidence
showing that individual rRNPs can be recycled for multiple
transcription and replication cycles. This is a key feature for
viral multiplication. In addition, their study highlights how
factors that affect the stability of the secondary structures of
the nascent RNA affect the rate of RNA synthesis.
The authors concluded that the approach is useful for
investigating viral transcription and replication mechanisms.
"Transcriptional pausing is an intrinsic property of most RNA
polymerases, and its regulation constitutes one of the central
mechanisms of control of gene expression," they add. "Future
single-molecule experiments of real-time RNA synthesis kinetics by
the IAV RdRp [influenza A virus RNA dependent RNA polymerase]
within the context of the RNP will help to elucidate the nature of
putative pause states and their roles in viral transcription and
replication."
Figure
https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Figure-1-2.jpg
Caption : Fig.1 (A, B) HS-AFM images of vRNP (A) and rRNP (B).
(C) Successive HS-AFM images of rRNP during RNA synthesis. © 2024
Carlero, et al. Published by American Chemical Society
Glossary
RNA synthesis
Ribonucleic acid (RNA) is a polymer and a nucleic acid. Its
replication is catalyzed by an enzyme known as RNA polymerase. The
synthesis only proceeds in the presence of the nucleotide and
proteins required to build up the RNA molecule.
Although RNA synthesis can use DNA as the synthesis template, a
number of viruses replicate with RNA as the template. The enzyme
that catalyzes this type of synthesis is known as RNA dependent RNA
polymerase.
Recombinant ribonucleic proteins
Recombinant ribonucleoproteins are useful for ways of studying
RNA processes. In this instance the authors used a recombinant
ribonucleoprotein made up of the same protein components but just
352 nucleotides long, where the RNA segment used had been shown to
avoid supercoiling.
High-speed atomic force microscopy
This imaging technique uses a nanosized tip at the end of a
cantilever that is scanned over a sample. It can be used to
determine the topography of a sample surface from the change in the
strength of forces between the tip and the sample with distance,
and the resulting deflection of the cantilever. It was first
developed in the 1980s but a number of modifications have augmented
the functionality of the technique since. It is better suited to
imaging biological samples than the scanning tunneling microscope
that had been developed because it does not require a conducting
sample.
In the 2000s Toshio Ando at
Kanazawa University was able to improve the scanning speed to such
an extent that moving images could be captured. This allowed people
to use the technique to visualize molecular processes for the first
time.
Reference
Diego Carlero, Shingo Fukuda,
Rebeca Bocanegra, Toshio Ando, Jaime
Martin-Benito, and Borja Ibarra Conformational Dynamics of
Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis
ACS Nano 2024.
DOI:10.1021/acsnano.4c01362
URL: https://pubs.acs.org/doi/10.1021/acsnano.4c01362
Funding acknowledgements
This work was supported by the NanoLSI Visiting Fellows Program
2019 (to B.I.), the World Premier International Research Center
Initiative (WPI), MEXT (Japan),
and grants PGC2018-099341-B-I00 (to B.I.), PID2021-126755NB-I00 (to
B.I.), and PID2020-117752RB-I00 (to J.M.B.) financed by
MCIU/AEI/10.13039/501100011033 and FEDER, UE, Grant
TED2021-132748B-I00 financed by the
European Union "NextGeneration EU"/PRTR (to J.M.B.), and Grant No.
20K15140 financed by JSPS KAKENHI (to S.F.). IMDEA Nanociencia
acknowledges support from the Severo Ochoa Program for Centers of
Excellence in R&D (CEX2020-001039-S).
Contact
Hiroe Yoneda (Ms)
Senior Specialist in Project Planning and Outreach
NanoLSI Administration Office, Nano Life Science Institute
(WPI-NanoLSI)
Kanazawa University
Kakuma-machi, Kanazawa 920-1192, Japan
Email: nanolsi-office@adm.kanazawa-u.ac.jp
Tel: +81 (76) 234-4555
About Nano Life Science Institute (WPI-NanoLSI), Kanazawa
University
Understanding nanoscale mechanisms of life phenomena by
exploring "uncharted nano-realms"
Cells are the basic units of almost all life forms. We are
developing nanoprobe technologies that allow direct imaging,
analysis, and manipulation of the behavior and dynamics of
important macromolecules in living organisms, such as proteins and
nucleic acids, at the surface and interior of cells. We aim at
acquiring a fundamental understanding of the various life phenomena
at the nanoscale.
https://nanolsi.kanazawa-u.ac.jp/en/
About the World Premier International Research Center Initiative
(WPI)
The WPI program was launched in 2007 by Japan's Ministry of Education, Culture,
Sports, Science and Technology (MEXT) to foster globally visible
research centers boasting the highest standards and outstanding
research environments. Numbering more than a dozen and operating at
institutions throughout the country, these centers are given a high
degree of autonomy, allowing them to engage in innovative modes of
management and research. The program is administered by the Japan
Society for the Promotion of Science (JSPS).
See the latest research news from the centers at the WPI News
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Main WPI program site:
www.jsps.go.jp/english/e-toplevel
About Kanazawa University
As the leading comprehensive university on the Sea of
Japan coast, Kanazawa University
has contributed greatly to higher education and academic research
in Japan since it was founded in
1949. The University has three colleges and 17 schools offering
courses in subjects that include medicine, computer engineering,
and humanities.
The University is located on the coast of the Sea of
Japan in Kanazawa – a city rich in
history and culture. The city of Kanazawa has a highly respected
intellectual profile since the time of the fiefdom (1598-1867).
Kanazawa University is divided into two main campuses: Kakuma and
Takaramachi for its approximately 10,200 students including 600
from overseas.
http://www.kanazawa-u.ac.jp/en/
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