2015 Joint Usage and Research Report
Title of Research Project

Structural and functional analyses of nuclear RNA granules inmammalian cells

ApplicantVisitingInstitution

Institute for Genetic Medecine

Job title

Professorand name

Tetsuro Hirose

Nameresearcher

Dr Gerard PIERRON, Research Director. Centre National de laRecherche Scientifique (CNRS) - France.

Purpose of the Research Project

Structural and functional analyses of RNA-dependent nuclear(approx. 250 words)

bodies in human cells.
Paraspeckle nuclear bodies are formed around an architectural
long non coding RNA, NEAT1, which is transcribed as two
distinct isoforms from a unique promoter. Our groups,independently, previously showed that both isoforms have a
distinct spatial distribution within the paraspeckles and adistinct role in their formation. The short isoform (3 700 nt) is
only found at the periphery of the bodies and is not sufficient for
their assembly when introduced into a NEAT1 KO cell. This
contrasts with the ability of the long isoform (22 700 nt), thecentral region of which is found within the bodies, to generateparaspeckles in NEAT1 null cells. Paraspeckles are uniqueamong

nuclearstructures

as

being

cylindricalwitha

constrained diameter (rather than roundish). We hypothesizedthat their shape and limited diameter was dictated by NEAT1 asa long, looped and folded transcript. In a collaborative study, we
further showed that, upon proteasome inhibition, transcriptionof both isoforms was enhanced, leading to paraspeckle expansion
by elongation with an unvarying diameter. In this setting, we
demonstrated that paraspeckles behave as “molecular sponges”,regulating gene expression by sequestrating regulatory factors.
Our research program is two fold: (i) create partial NEAT1
deletion mutants by CRISPR/Cas9 technology and analyze athigh resolution the effects that are resulting on the assembly,
the ultrastructure, the size and the function of the paraspeckles(ii) define ultrastructurally nuclear bodies newly-identified in
Sapporo, that contain essential RNAs and protein components(some being cancer-related factors) and that, depending on thecellular context, are seen as fused or separate entities. Thissecond project is technically reminiscent of our recent

demonstration

oftheformation

byfusionofhybridPML/p62-SQSTM1 bodies in Hela cells by electron microscopy(Souquere S. et al, Nucleus 6:326-38, 2015). Defining theinteractions between these nuclear bodies will help todiscriminate between different models of nuclear body formation
likeself-organization(randomorderofassociationofcomponents), liquid droplet behavior or ordered assembly withdefined molecular seeds.
Development of the Research

We have carried out a series of collaborative experiments since

Project and Results2012 leading to major progresses in paraspeckle knowledge. In(approx.. 850 words)

short, combining molecular and microscopic analyses we havedemonstrated the exquisite sensitivity of the NEAT1 lncRNAand of the paraspeckles towards proteasome inhibition. This
provided us with a paradigm of paraspeckle expansion,
enhancinggreatly

theirfunction

sothathalfofthenucleoplasmic content of major regulatory factors such as
NONO and SFPQ was displaced and sequestrated in theparaspeckles. This resulted in major changes in gene expression
as measured by us and by another group (with another inducerof paraspeckle expansion). We also identified the SWI/SNF
chromatin-remodeling complex as a surprising paraspeckle
assembly factor. This result was a direct consequence of theidentification by the Hirose’s group of 35 paraspeckle proteincomponents, a number of which turn out to be classified as
SWI/SNF interacting proteins in proteome databases. As afollow up, it was shown that silencing of both BRG1 and BRM
SWI/SNF ATPases was leading to paraspeckle disintegration.
Disentangling interactions between essential assembly factors
like the lncRNA NEAT1 and essential protein components suchas NONO, SFPQ, SWI/SNF is crucial to implement ourunderstanding of the molecular interactions at play inparaspeckle assembly.
During my visit at the Institute of Genetic Medicine of theHokkaido University in Sapparo (August 19-September 10,2015), at the invitation of Professor Tetsuro Hirose, we initiateda series of collaborative experiments, combining molecular andstructural approaches, like analysis of NEAT1 deletion mutantsby immuno-fluorescent microscopy, super resolution microscopy
and immunogold electron microscopy. Various partial deletionmutants of the long NEAT1 isoform were created in Sapporo andmutated cell lines scrutinized under the EM to pinpoint
resulting changes on paraspeckle morphology. HAP1 cells havebeen used because of their haploid chromosome content. We
defined paraspeckle geometry in this cell type and we checkedthat they were sensitive to proteasome inhibition. In doing so,
we have noticed the formation of membrane-less cytoplasmic
bodies that contain a variety of paraspeckle proteins. Since someparaspeckle components such as FUS or TDP43 are prone, incertain circumstances, to generate pathogenic cytoplasmic
aggregates, we are further analyzing the stress conditions
leading to the formation of these cytoplasmic “bodies” and theirrelationship, if any, to ubiquitous membrane-less cytoplasmic
structures like the P-bodies.
Our results confirm that the long NEAT1 isoform is essential forparaspeckle assembly and reveal that the paraspeckle diameteris greatly reduced by the deletion of central regions of thislncRNA. In doing so, we have defined the genetic determinants
that are shaping the paraspeckles, with a length depending on
NEAT1 abundance and a diameter determined by NEAT1 size.
In parallel, we have defined ultrastructurally, by immunogold
electron microscopy, one of the 2 nuclear bodies that are seen as
separate entities in the U2OS osteo-carcinoma derived cell line
and as a single body in cervical cancer derived HeLa cells. In theabsence of an efficient antibody to characterize the second bodyunder the electron microscope, we used GFP-fusion proteins asmarkers. However, weare

facing unpredicted

technical

difficulties in this project. It is still not yet clear whether
chemically-fixed cell samples were “damaged” by transportation
between Japan and France or whether there is an intrinsic
difficulty as is occurring when GFP epitopes are masked by thefused protein and therefore not accessible to antibodies. We areactively pursuing this study that will help to characterize thedynamic relationship of these bodies in tumoral cells.
Recent reports are also suggesting that NEAT1 and theparaspeckles are markers and potential prognostic factors forcancer development. Our studies will improve our knowledge ofthe functional and structural properties of cancer cells.
Our most recent results are not yet published but will
likely be in the next few months.

Publication【Conference, symposium, workshop etc.】*Enter the names of conferenceor journal and its vol. No. where
the above work was presented.

【Journals】
Kawaguchi T, Tanigawa A, Naganuma T, Ohkawa Y, Souquere S, Pierron G,Hirose T. SWI/SNF chromatin-remodeling complexes function in noncoding
RNA-dependent assembly of nuclear bodies. Proc Natl Acad Sci U S A. 2015;
112: 4304-9.

T. Hirose, G. Virnicchi, A. Tanigawa, T. Naganuma, R.Li, H. Kimura, T. Yoko
S. Nakagawa, M. Benard, A. Fox, and G. Pierron
NEAT1 long noncoding RNA suppresses transcription via protein
sequestration within subnuclear bodies. Mol Biol Cell 2014; 25:169-83.