2016Joint Usage and Research Report
Title of Research Project

Functional interpretation of the molecular interaction between
long noncoding RNA and RNAi machinery in the nucleus

Institution
ApplicantJob title andname
Name

Professor Valerio ORLANDO

Visiting

Head of KAUST Environmental Epigenetics Program

researcher

KAUST, Thuwal Saudi Arabia and Santa Lucia Foundation,
Rome Italy

Purpose of the Research Project(approx. 250 words)

The key research topics of the collaboration are:1.

Role of nuclear RNAi components in gene expression regulationand chromatin architecture

2.

Investigating the functional association of RNAi components withlncRNAs and their role in the assembly of nuclear bodies called
paraspeckles

3.

Understanding the role of lncRNAs (NEAT-1 and MALAT-1) in
3D genome organization

Development of the Research

2. Research Project

Project and Results

The role of RNAi in post-transcriptional gene silencing in the cytoplasm is(approx.. 850 words)

well characterized. Although, nuclear RNAi proteins (Argonaute-1 andDicer) and long non-coding-RNAs (ncRNA) have shown to be enriched in
the chromatin, however, little is known about their collective role in the cellnucleus and the mechanisms that regulate ncRNA function. The goal of thisproject is to investigate the nuclear functions of RNAi components andlncRNAs in mammalian cells, particularly in nuclear architecture dynamics
in somatic cell response to environmental stress. A combination of genomics,
protein complex biochemistry and imaging approaches are used.
Progress
To date, we have identified a novel role for nuclear Argonaute-1 in 3D
genome organization and gene expression regulation in human cells. In thisstudy, I discovered for the first time that nuclear Argonaute-1 associates withlong-coding RNA and regulate 3D genome structure. To our knowledge thisis a breakthrough in the field of 3D genome biology and will further ourunderstanding of the complexity of genome structure and function both innormal development and diseases. We presented this work in a Keystone
symposium “Noncoding RNAs: From Disease to Targeted Therapeutics" at
the Fairmont Banff Springs, Banff, Alberta, Canada. And a high impact
journal manuscript “ A novel role of nuclear Ago1 in regulating chromatinarchitecture and gene expression” Muhammad Shuaib, Krishna Mohan Parsi
et al., 2017 is in preparation.

Interestingly, we discovered another important function of nuclear RNAicomponents (Ago1 and Dicer1) in the assembly of NEAT-1-lncRNA
containing nuclear bodies called paraspeckles. Paraspeckle nuclear bodies
play important roles in various nuclear processes such as gene regulation andnuclear organization. A researcher from Orlando’s lab, Dr. Muhammad
Shuaib, visited Prof. Hirose’s laboratory for three weeks (2016) to identify
the functional association of RNAi components with NEAT-1- lncRNA andparaspeckle by using RNA-FISH. During this visit he successfullycompleted all the experiments and obtained very interesting results thatcomplement our research project at KAUST. This work is under preparation
for a second manuscript that we will publish soon in a well-reputed journal;“Chromatin associated RNAi components function in the assembly of
NEAT1 lncRNA-containing paraspeckle. Muhammad Shuaib et al.,”Moreover, we have identified a novel previously uncharacterized isoform of
human Dicer that is highly enriched in the nucleus. we found a short uniquepeptide at the N-terminal of this protein, which we used for the generation ofcustom-made antibody. We have used this antibody for the characterizationof this short isoform in various cell lines. The preliminary results indicatethat this protein is present in different human cells, suggesting its essential
role in the nucleus.

Another collaborative project initiated with the group of Prof. Hirose is toinvestigate the function of long-non-coding RNAs (NEAT1 and MALAT1)
in chromatin architecture by using high through-put chromatin conformation
analysis (Hi-C). I used NEAT1, MALAT1 and double knockout cell lines thatwere obtained from the laboratory of Prof. Tetsuro, for the Hi-C experiments.
We successfully completed the construction of all Hi-C libraries and obtained
good quality data after sequencing in the KAUST core lab. The data is now
available for further analysis and integration with other data sets, in order tocomplete the project.
To further understand the mechanism and function of nuclear RNAicomponents (Ago1 and Dicer1) we are using an integrated proteomic
approach to decipher in vivo protein-protein interactions and applied thisstrategy to globally map the Ago1 and Dicer1 interaction network in humancells. This project involves the generation of stable cell lines expressing
FLAG-HA-tagged RNAi components (Ago1 and Dicer1) proteins. By
tandem affinity purification (TAP-TAG) and mass spectrometry analysis I
have identified key components of paraspeckle nuclear bodies associatedwith Ago1 in chromatin fraction.
Furthermore, to study the nuclear localization and co-localization of RNAicomponents, we applied the genome-editing technology (CRISPR-CAS-9)
for tagging of endogenous genes (Ago1 and Dicer1) with GFP. We generated
stable HeLa cell lines expressing GFP-tagged endogenous Ago1.

Remaining Work and Expected Results

1) To complete the collaborative project with the group of Prof.
Hirose, which involves investigating the function of long-noncoding RNAs (NEAT1 and MALAT1) in chromatin architecture.
This project aims to explore a novel link between nuclear lncRNAs(NEAT1 and MALAT1) and chromatin associated RNAicomponents in regulating 3D genome structure. This work will leadto another high impact manuscript.

2) To fully understand the function of above mentioned newly
identified short isoform of human Dicer, by CRISPR-mediateddeletion, CHIP-seq and RIP-seq. The results from this work will
clarify the nan-canonical role of nuclear Dicer and might open new
avenue for future research.

Publication

Noncoding RNAs: From Disease to Targeted Therapeutics (J5)

*Enter the names of conference orFebruary 5—9, 2017

journal and its vol. No. where theFairmont Banff Springs • Banff, Alberta Canada

above work was presented.

【Journals】