1. Introduction
We are delighted to share with you our sixth Journal Club and highlight some of the
most interesting papers published recently. We hope to keep you up-to-date with non-coding
RNA research works that are outside your study area. The Non-Coding RNA Scientific
Board wishes you an exciting and fruitful read.
2. Special Delivery from Plants to Pathogens
Highlight by Hua Xiao and Patrick K. T. Shiu
Although it is known that hosts can send small RNAs (sRNAs), to pathogens, to inhibit
their virulence, the mechanism of this transfer is unclear. In a recent issue of Science,
Qiang Cai and coworkers showed that extracellular vesicles are involved in the process
[1].
In this study, the authors purified fungal (Botrytis cinerea) cells from infected
Arabidopsis tissues and identified 42 host-originated sRNAs (most of which could also
be detected in extracellular vesicles isolated from apoplastic fluids). In animals,
microRNAs are transferred via exosomes, which are derived from multivesicular bodies
(MVBs). Tetraspanins (TET) are exosome markers in mammals, and Arabidopsis TET8 accumulates
at the infection sites. At these sites, MVBs fuse with the plasma membrane to release
vesicles (which can be readily uptaken by fungal cells), further supporting the notion
that plants secrete exosomes to deliver sRNAs to their pathogens. Half of the transferred
sRNAs have predicted targets, with a bias towards vesicle-trafficking pathways (which
are important for fungal virulence). The target genes are down-regulated after an
infection, unless the plant host is deficient in trans-acting small interfering RNA
(tasiRNA) production. The cleavage of mRNAs could be detected in the fungus, suggesting
that the transferred sRNAs silence the target genes through a transcript destruction.
This work showed that plant sRNAs are delivered to fungal cells via pathogen-induced
exosomes. Future studies should shed light on how these defensive sRNAs are selectively
packaged and transported and if this weapon delivery system is universal in cross-kingdom
RNA interference (RNAi).
3. Connecting Myofibroblasts and Cardiomyocytes via Extracellular Vesicle-Associated
microRNA: A Short-Distance Affair
Highlight by Jun Shu and Gaetano Santulli
The expression of the small ubiquitin-like modifier 1 (SUMO1) has been reported to
be significantly reduced in both human and murine heart failure (HF). However, the
molecular mechanisms underlying SUMO1 upregulation had not been investigated, hitherto.
Changwon Kho and colleagues identified a specific microRNA (miR) as a key factor in
the pathophysiology of HF [2]. The authors demonstrated that miR-146a is a SUMO1-targeting
miR, in failing human and mouse hearts. In a model of pressure overload, overexpression
of miR-146a reduced SUMO1 expression, whereas miR-146a inhibition normalized SUMO1
expression and improved the cardiac function. Intriguingly, the authors demonstrated
that miR-146a was not directly produced by cardiomyocytes, but it was first synthesized
by activated fibroblasts (myofibroblasts), following an injury, and then transferred
via extracellular vesicles to cardiomyocytes. This discovery has major implications
in the clinical scenario, since targeting miR-146a might provide a novel therapeutic
approach for the treatment of HF.
4. Size-Dependent Export of Circular RNAs from the Nucleus
Highlight by Mohammad K. Gheybi and Simon J. Conn
While circular RNAs are co-transcriptionally synthesized in the nucleus, they are
almost exclusively sequestered into the cytoplasm. This suggests circular RNAs (circRNAs),
like other RNAs, are actively exported from the nucleus. In a recent issue of Genes
& Development, Chuan Huang and co-workers illuminated a size-dependent mechanism of
nuclear transport for circRNAs [3].
Using a targeted, small-interfering RNA (siRNA) screen against proteins involved in
nuclear RNA export in Drosophila, the authors identified an RNA helicase—Hel25E—which
caused nuclear retention of nascent circRNAs, longer than 800 nt. However, depletion
of the principal mRNA nuclear export factor, the NXF1:NXT1 complex, had no quantifiable
effect on circRNA transport. Extrapolating these results to human cells, targeting
the two Hel25E homologs in HeLa cells, URH49 (DDX39A), and UAP56 (DDX39B), resulted
in a nuclear retention of short (<356 nt) and long (>1298 nt) circRNAs, respectively.
This study identified the catalytic ATPase and helicase domains, of these human RNA
helicases, as critical factors in defining circRNA export-size preference. Future
studies could illuminate factors contributing to the nuclear export of intermediate-sized
circRNAs and the possible involvement of distinct nucleoporins within the nuclear
pore complex.
5. A New Bifunctional RNA at the Origin of Tumor Metastasis
Highlight by Baptiste Bogard and Florent Hubé
PNUTS (Phosphatase 1 Nuclear Targeting Subunit) was originally identified as a modulator
of the PP1 phosphatase on Retinoblastoma protein (Rb). PNUTS is up-regulated in cancer
cells where it acts as a potential oncogene through sequestration of the tumor-suppressor
PTEN in an inactive state, in the nucleus.
Grelet and colleagues identified an alternatively spliced isoform of PNUTS mRNA generated
through an alternative acceptor splice site, in exon 12. The resulting frame-shift
disrupted the translation capacity of the transcript, hence producing a long non-coding
RNA (lncRNA-PNUTS) [4]. The authors identified heterogeneous nuclear ribonucleoprotein
(hnRNP) E1 as being required to prevent alternative splicing of PNUTS precursor RNA
and favor the formation of the PNUTS mRNA. They showed that the lncRNA-PNUTS acts
as a sponge for miR-205, a negative regulator of genes involved in epithelial-to-mesenchymal
transition (EMT), limiting its bioavailability. They tested the consequences on tumor
progression and showed a temporary increase in the expression of the ZEB proteins,
a family of transcription factors that regulate EMT, which in turn trigger downstream
EMT events.
This study reported an example of a mammalian lncRNA—produced by an originally protein-coding
gene—through alternative splicing, which is involved in the early stages of EMT and
possibly cancer progression.
6. Screening for Long Non-Coding RNA Mediators of Chemotherapy Resistance
Highlight by Joseph H. Taube and Sendurai A. Mani
Forward genetic screens have yielded fantastic insights into gene-phenotype relationships,
for decades. Bester and colleagues have adopted this strategy to identify long non-coding
RNAs (lncRNAs), as well as protein-coding genes, which, when over-expressed, confer
altered sensitivity to Ara-C, a deoxycytidine analog used to treat acute myeloid leukemia
[5]. As lncRNAs exert their function through both trans and cis mechanisms, endogenous
loci, rather than transgenes, were activated using a CRISPRa-SAM [6]. This was achieved
by linking a deactivated Cas9 to the VP64 co-activation domain, expressing a synthetic
co-activator, and then applying a library of aptamer-tagged single guide RNA (sgRNA)
specific to the known lncRNA and coding gene promoters.
The authors identified hundreds of candidate lncRNAs, either associated with Ara-C
sensitization, or resistance. They further confirmed the role of several lncRNA, including
the GAS6 antisense transcript (GAS6-AS2). GAS6-AS2 was further shown to act in cis
mechanisms to upregulate the expression of its cognate protein-coding gene (GAS6),
and in trans mechanisms to alter the DNA methylation at AXL. Validating this strategy,
a higher expression of GAS6-AS2, as well as AC008073.2, were associated with poor
prognosis and decreased disease-free survival, in acute myeloid leukemia (AML) patients
treated with Ara-C. This approach showed the power of forward genetic approaches to
capture key regulators among lncRNA-encoding genes.
7. Long Non-Coding RNA “Entrap” Tumor Suppressor microRNAs to Promote Cancer Growth
and Migration
Highlight by Luo Song, George A. Calin and Shuxing Zhang
We have a deep interest in modeling RNA–protein [7], RNA–RNA [8], and RNA–small molecule
interactions [9]. Such profundity will significantly help us elucidate novel molecular
mechanisms of tumorigenesis and provide insights for therapeutics discovery and development.
Herein, we are delighted to share one of the interesting papers we published recently
on ncRNA–ncRNA interactions [8].
The transcribed ultraconserved regions (T-UCRs) had been proven to play an important
role in human carcinogenesis, but there is still a large blank in the mechanisms and
the consequences of their expression dysregulation in cancers. Recently, our colleagues
revealed a new pathway of the transcribed ultraconserved region 339 (uc.339) to explain
how uc.339 promotes carcinogenesis [8].
In this article, the authors assessed the related data from The Cancer Genome Atlas
(TCGA) database and found that, in 210 non-small cell lung cancer (NSCLC) patients,
the high expression of uc.339 was positively correlated with the low survival. Furthermore,
the authors showed that with upregulation of the transcribed uc.339 in archival NSCLC
samples, tumor suppressor microRNAs, such as miR-339-3p, -663b-3p, and -95-5p, were
suppressed, which led to an increased level of a cell-cycle regulation protein, cyclin
E2. As a result, cancer growth and migration was promoted. Additionally, the result
suggested that either overexpression or suppression of these microRNAs had limited
effects on the uc.339 level. Based on their modeling studies, the authors named this
type of interaction as “entrapping”. This study further revealed structural details
of how lncRNAs play roles in human carcinogenesis, and also provided a novel concept
for cancer therapy and drug discovery.