The manifestation of the pluri- and multipotency

The
totipotency of the germline is the full manifestation of the pluri- and
multipotency of embryonic and adult stem cells, thus the germline and stem
cells must share common mechanisms that guarantee their multipotential in
development. During embryogenesis the lifecycle of mRNAs relies on the length
of poly(A) tails, which is variable and regulates translational efficiencies
which mark specific transitions during development. A similar correlation
between poly(A) tail length and translational efficiencies was recently
correlated with the progression of cell cycle in somatic cells. During early
development, differential mRNA stability plays a critical role in the
regulation of gene expression. Poly(A) tails are essential 3′
post-transcriptional modifications of eukaryotic mRNAs and their length defines
timing for important stages of mRNA lifecycle such as post-transcriptional
quality control, translational rate and decay 1. They are dynamically shaped
by the opposing effects of poly(A) polymerases and deadenylases, a specific
class of exo-ribonucleases. The existence of several and diverse deadenylases
with distinct features highlights the significant mediation of these enzymes in
essential cellular functions. Moreover, eukaryotic genomes contain putative deadenylases
with elusive, so far, biological role. In addition to their prominent mRNA
targets, several deadenylases are also involved in the maturation of important
small and long noncoding RNAs, an observation which elaborates their essential
role in regulating gene expression at various levels 2. As we move up in the
evolutionary ladder, the number of deadenylases increases significantly, an
observation which suggests that although all deadenylases catalyse poly(A)
removal, they can mediate additional reactions, in complex with auxiliary
proteins which alter their substrate repertoire 4. Deadenylases can be
divided into two major superfamilies based on the topology and properties of
their active sites the DEDD family (CAF1, PAN2, PARN and PNLDC1) and the EEP
family (CCR4, Nocturnin, ANGEL and PDE12). In mammals, CCR4-CNOT complexes and,
in many cases, PAN2-PAN3 complex, are the major deadenylases responsible for
mRNA turnover and mediate maternal mRNA clearance during Maternal to Zygotic
Transition (MZT), in senescence, in DNA damage response and cell cycle control,
neuronal development and in the generation of induced iPSCs 4,5. In addition,
CNOT3, a subunit of the CCR4-NOT deadenylation complex is an essential factor
for stem cell renewal. The elevated expression of ribonucleases during these
early stages of development may act as mediators of a specific translational
program that leads to the establishment of the different cell lineages. It has
been suggested that deadenylases are required for maintaining the pluripotent
state of stem cells, in association with RNA-binding proteins that fine-tune
the development of the early embryo 6.

Recently we were the first to identify and
characterize the human and mouse PNLDC1 gene Poly(A)-specific ribonuclease
(PARN)-like domain containing 1, which encodes for a novel and highly specific
deadenylase. Surprisingly, PNLDC1 is exclusively expressed in mouse embryonic
stem cells (mESCs) and spermatocytes 4. During the same period, PNLDC1 from
B. mori was reported as the trimmer enzyme responsible for pre-piRNAs
maturation, an observation which was confirmed recently in mouse spermatocytes
(XX). Although mRNA deadenylation is catalysed by PNLDC1 without requirements
for auxiliary proteins, the unexpected involvement of PNLDC1 in pre-piRNA
maturation requires association with Piwi proteins loaded with pre-piRNAs.
Recent studies indicate that the Piwi-piRNA pathways mediates epigenetic
reprogramming and poststranscriptional regulation, which may be responsible for
its functiuon in germline specification, gametogenesis, stem cell maintenance,
transposon silencing and genome integrity in diverse organisms. Intriguingly,
PNLDC1 expression is under epigenetic regulation by DNMT3B methyltransferase an
important enzyme involved in the differentiation of embryonic stem cells and in
silencing of genes implicated in differentiation and carcinogenesis. Notably,
PNLDC1 expression is undetectable in differentiated cells, and is induced only
after treatment with the demethylating agent 5-AZA-CdR, a known anticancer
drug. Our observation that PNLDC1 is specifically expressed in spermatocytes
and mESCs pinpoints the exquisite role of PNLDC1 9,10. Both embryonic stem
cells and germline share common mechanisms to maintain multipotency and
therefore PNLDC1 poses as a key-enzyme that could regulate specific mRNA
turnover and clearance, as well as maturation of essential non-coding RNAs such
as piRNAs, lncRNAs and many snoRNAs. Preliminary transcriptomic analysis that
we performed after effective silencing of Pnldc1 in mESCs verified our
hypothesis and showed significant modulation of genes that shape cell cycle,
DNA replication repair, chromatin remodeling, transcription modification and
translational regulation. Moreover, Pnldc1 silencing affects the transcriptomic
profile of pre-piRNAs compared to mature piRNAs, a finding that verifies the
existence of piRNAs in mammalian ESCs and poses questions on their role. In
addition, many essential lncRNAs such as the RNA subunits of nuclear and
mitochondrial RNase P, DANCR, Malat1 and Meg3 were significantly affected. Long
non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the
self-renewal in human embryonic stem cells (hESCs) 11. An increasing number
of studies demonstrated that lncRNAs modulate stem cell biology by interacting
with essential transcription factors responsible for maintaining pluripotency
or regulating differentiation 12. All these events occur also during germline
biogenesis and depend on dynamic processes from a very complex molecular
landscape, which awaits further investigation. Therefore, the role of PNLDC1
beyond mRNA turnover and especially on the biogenesis of noncoding RNAs in
mESCs will provide new insights on the regulatory role of deadenylases in
higher eukaryotes and especially in mammals. Finally, gene networks that
activate deadenylases are essential and therefore targeting of deadenylation
has been proposed as a new anti-cancer strategy based on the fact, that
proliferating cells require continuous and elevated production and recycling of
specific mRNAs. Available crystal structures from few known deadenylases have
provided knowledge on their specificity and function, however this knowledge is
still limited, and more structures are needed. The ability to modulate
deadenylase activity with novel ligands is important as we have previously
shown for PARN deadenylase 15, 16. Finally, it requires structural
characterization and determination of the 3D structure of more representative
deadenylases, like PNLDC1, which will allow the functional determination of
protein-protein and RNA-protein networks that are essential for gene expression
regulation. 

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