Abstract and 5sRNA gene transcription, although, SIRT7 overexpression did

Abstract

Localization of Sirt7
in nucleolus indicated that it is involved in rDNA transcription and ribosome
biogenesis. In humans, the rRNA genes are located on five different chromosomes
(13, 14, 15, 21 and 22) all of which loop to form nucleolus and are
subsequently transcribed by RNA polymeraseI. SIRT7 was found to be associating
with RNA POLI for rDNA transcription. Studies also showed that SIRT7 interacts
with various components of TFIII2 complex and components of RNA POLIII complex involved
in tRNA and 5sRNA gene transcription, although, SIRT7 overexpression did not
increase protein synthesis indicating its indirect involvement in protein
synthesis. The ribosomal protein synthesis gets affected by Sirt7 and RNA POLIII
association and dissociation. Earlier study results clearly suggest that SIRT7
is involved in regulating both POLI- and POLIII-dependent transcription. Here
we present a detailed review report of SIRT7 involvement in rDNA transcription
and protein synthesis and its role in cellular stress.

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Introduction

Epigenetics has now become the most studied area as
it is seen to be involved extensively in gene regulation. It is widely accepted
by the living system because of its ability to be reversed and can be
transferred to next generation. Epigenetics constitutes post translational
modification as the main machinery for regulating gene expression among which
DNA methylation, chromatin remodeling and histone acetylations /deacetylations
are some of the extensively used mechanisms, histones are the major target of
these processes for both repressing and activating a particular gene to carry
out a molecular process. Histone deacetylases (HDAC’s) are the enzyme which
carry out deacetylations of acetylated histones by removing acetyl groups from
an
?-N-acetyl lysine amino acid on a histone making it more compact and unavailable for
any molecular process. Sub-families of HDAC are class1-class4 and here we will
concentrate on a class3 HDAC called sirtuins7. Unlike all other HDAC’s sirtuins
has a  NAD+ binding catalytic domain and
widely distributed in nucleus (SIRT1,2,6,7), cytoplasm (SIRT1,2), mitochondria
(SIRT3,4,5).Amongst all the sirtuins sirt1 and sirt7 is involved in various
metabolic and cellular functions like energy metabolism, DNA damage, regulates
fat oxidation in liver, ageing in lower vertebrates, in higher vertebrates
inspite of directly involved in ageing regulation it is regarded as a metabolic
factor. Its localization in nucleolus indicates its involvement in ribosome
biogenesis or protein synthesis and this has been proved by various researchers
because ribosomes are found in close proximity with the nucleolar region.

Sirt7 is located on
chromosome 17 in subtelomeric region with 10 exons and an ORF of 1203bp
encoding a protein length of 400 amino acids. It has a molecular weight of 44KD
and its catalytic domain is present between 90 and 331 amino acid residues. In
normal conditions it is highly expressed in metabolically active tissues like
CD-33 positive myeloid bone marrow precursor cells, liver and almost all
tissues except non proliferating tissues like muscle, brain and ovary. Like all
other sirtuins Sirt7 has a weak deacetylase activity, some of the known
substrates are p53, h3k18ac, NPM1, PAF53,GABPbeta1.

 

table1. Showing
Interacting partners and substrates of SIRT7

 

Our focus here is
PAF53 substrate the deacetylase activity of SIRT7 is also involved in rDNA
transcription. RNA polymerase1 is responsible for rDNA transcription which is
required for synthesis of ribosome. Metabolically active cells are in need of
continuous ribosome synthesis and function for which Sirt7 deacetylates PAF53
(at position k373) a subunit of RNA polymerase1 this activity of SIRT7 is
required for RNA pol1 binding to rDNA promoter for transcription initiation and
elongation2.

 

SIRT7 and rDNA transcription

Ribosomal RNA is
synthesized in nucleolus and the genes for this RNA is clustered in five
different chromosomal tips found in nucleolus3.SIRT7
is found to be closely associated with rDNA promoter along with RNA polymeras1
and this was proved when RNase treatment caused redistribution of Sirt7 from
nucleoli to nucleoplasm indicating association of SIRT7 with RNA plymerase1
during transcription in nucleolus. Co-immunoprecipitation assays involving
antibodies against RNA polymerase1 but not control antibodies co-precipitated
endogenous SIRT712.Similarly, when RNA Polymerase1 was
co-precipitated with alpha FLAG(M2) antibody against FLAG tagged SIRT7 clearly
indicating that SIRT7 interacts with RNA polymerase1 machinery to carry out
transcription12. Knockdown of SIRT7 caused significant
decrease in rRNA synthesis followed by lesser association of RNA polymerase1 to
the rDNA promoter and overexpression caused increase in RNA ploymerase1
mediated transcription4. This clearly indicates functional role of
SIRT7 in rDNA transcription but clear functional characterization of SIRT7 is
not specified.

As we know histone
deacetylase do not have direct DNA binding domain so they need an additional
protein with a DNA binding domain. Hence, It has been reported that
UBF(upstream binding factor) which binds to the upstream region of rRNA genes
and recruits other factors required for forming pre-initiation complex like
SL1, SL1 consists of TATA binding protein(TBP) and TAF’s.This association was
more clear when sertinol an inhibitor of sirtuins caused inhibition of rDNA
transcription.5.It is reported that SIRT7 is found
associated with UBF in most cell cycle stages(G1, G1/S, Sphase) and even during
mitosis SIRT7 and UBF are complexed and remain bound to chromatin instead of
residing in NOR’s.

As discussed above
SIRT7 has a direct deacetylating substrate PAF53 which is a component of RNA
polymerase1 complex this association of SIRT7 and PAF53 is essential for
recruitment of RNA polymerase1 on rDNA promoter by interacting with other
subunits and UBF6. Co-immunoprecipitation experiments
confirmed the association of SIRT7 and PAF53.The association of PAF53 with RNA
POLI was not affected by both wild type and mutated PAF53(K373) suggesting that
acetylation does not affect interaction of PAF53 with POLI.Mutated
PAF53(arginine in place of lysine) revealed high pre-rRNA synthesis indicating
hypoacetylation is important for rRNA synthesis6.During
stress, when SIRT7 is released from nucleoli the association of PAF53 with
SIRT7 and subsequently with RNA polymerase1 is lost due to hyperacetylation of
PAF53 and thereby halting rDNA transcription6.Pre-rRNA
synthesis was reduced in both cases when CBP and PAF53 was knocked down
separately suggesting that both acetylation and deacetylation by SIRT7 is
necessary for rRNA synthesis but PAF53 occupancy was slightly increased in CBP
depleted cells.

During stresses
like glucose deprivation or due to some treatment when SIRt7 is released from
nucleoli rDNa transcription is halted and hyperacetylation of PAF53 is seen
whereas in mutated PAF53 (K373R) no acetylation was observed indicating that
K373 is the only lysine residue which is deacetylated during stress. Perturbed
interaction between PAF53 and SIRT7 was seen during stress which led to
hyperacetylation of PAF53 but the association of PAF53 with RNA POLI was
affected only a little by 30% but not to a great extent contradicting the fact
that deacetylated PAF53 is required for RNA POLI and rDNA association.Treatment
of cells with RNAse A showed that binding of SIRT7 and PAF53 depends upon RNA
because SIRT7 and PAF53 interaction was abolished.On the other hand this RNAse
A treatment did not affect UBF and rDNA interaction whereas SIRT7 was released
from nucleoli indicating that SIRT7 protein-protein interaction and its
retention in nucleoli is dependent on RNA.

Another interacting
partner of SIRT7 involved in rDNA transcription is RPA194(table1) the largest
subunit of RNA polymerase1.Anti-RPA194 affinity resin incubation with
transcriptionally competent cell extract showed failure in transcribing rRNA
genes. This subunit is involved in elongation process of transcription as it
helps in movement of RNA POLI enzyme complex three bases ahead during
elongation this characteristic feature became more clear when RPA194, RPA127,
RPA40 and PAF53 eluted in the same immunoblots but UBF(involved in
transcription initiation) co-elution was not found7.Although functional characterization of RPA194 is still not done but
knockdown of SIRT7 by siRNA in hela, A549, U2OS,ZR75 cells showed significant
decrease in the protein level of RPA194 but deacetylation activity of SIRT7 on
RPA194 is yet not stated8.Co-localisation of SIRT7 and RPA194 was not
hampered by RNAse treatment suggesting SIRT7 and RNA POLI participate in tight
interaction.

Sirt7
and chromatin remodeling complexes

According to the
proteomic analysis data of Tsai et all SIRT7 is found to interact with the
components of B-WICH chromatin remodeling complexes like WSTF, Mybbp1A, SNF2H,
etc. According to the STRING database results all the components of B-WICH and
other chromatin remodeling complexes are found to be associated with SIRT7 but
the direct interactions and functional link between these proteins were still
missing. To get more clear picture of SIRT7 functionality the  number of associated partners were  narrowed down by checking the abundance of
the protein in immunoisolations alongwith SIRT7 and calculating the NSAF values
which were further normalized by PAX database14.The
results showed that the components of B-WICH were highly abundant for example
SMARCA2, SMARCA4, RSF1, BAZ1B, BAZ2A, TTF1 these are the proteins involved in
remodeling of the chromatin regions involved in rRNA synthesis. Since this data
does not provide the clear picture of SIRT7 involvement in chromatin remodeling
by direct interaction with the components it could be associated with B-WICH
via some other protein which needs to be addressed.

 

SIRT7 and protein synthesis

SIRT7
like yeast Sir2 is localised in nucleoli which is the center for ribosome
biogenesis SIRT7 is involved in protein synthesis also as its knockdown in
liver cancer cells showed decrease in the level of ribosomal proteins9.SIRT7 was found to be associated with
TFIIIC2 components in affinity purification and mass spectrometry analysis.When
SIRT7 was found interacting with RNA polymerase III components (POLR3B, POLR1D,

POLR3A, POLR1C, POLR3C, POLR3D,
POLR2H and POLR2E) and TFIIIC2 complex (GTF3C1, GTF3C2, GTF3C3, GTF3C4, GTF3C5
and GTF3C6) 8.Since
ribosome are the translational machinery and RNA polymerase III is involved in
tRNA and 5srRNA SIRT7 knockdown loses its association with the above mentioned
translational components thereby halting translation of ribosomal proteins. On
the contrary, SIRT7 overexpression did not increase ribosomal protein synthesis
indicating an indirect effect of it on translation but SIRT7 overexpression
caused decrease in the level of ribosomal protein RPS7 and RPS14, this may be
mediated though MYC regulated SIRT7 association to ribosomal protein genetic
regions to decrease ER stress 10.

Knockdown of SIRT7 caused decrease in both protein
synthesis and rDNA transcription but inhibition of protein synthesis was
preferred over rDNA transcription.8. In order to
determine the involvement of SIRT7 in protein synthesis interaction between
SIRT7 and RPL11 a protein found in 60S subunit was investigated and the result
showed that SIRT7 and RPL11 show co-localisation in monoribosomal regions and
are palying an important role in cell cycle progression.SIRT7 knockdown
resulted in release of RPL11 to polyribosome region and subsequent silencing of
SIRT7 by siRNA and to study its effect in protein synthesis by measuring the level
of amino acid analog HPG incorporation showed that SIRT7 is necessary for
protein synthesis.

Although SIRT7
knockdown preferentially inhibits protein synthesis over rDNA transcription
SIRT7 overexpression did not increase protein synthesis significantly
indicating that SIRT7 is involved in regulating protein synthesis via various
pathways and not through a single pathway.

SIRT7 regulates
protein synthesis by interacting with mTOR and TFIIIC2 forming a complex and
affecting the function of RNA POLIII.mTOR is a nucleolar protein and its localization
there alongwith SIRT7 suggests its efficient involvement in protein
synthesis.After all these studies and confirmation about SIRT7 involvement in
protein synthesis and ribosomal biogenesis it was essential to study its
deacetylation activity on the interacting partners for which v global acetylation
levels of nucleolar proteins were measured before and after SIRT7 knockdown and
the results showed that there was not much effect on the levels of acetylation
globally before and after treatment however SIRT7 had protein and tissue
specific deacetyalase activity which may be responsible for its unique function
in regulation of rDNA transcription and protein synthesis.Immunoaffinity
studies revealed that SIRT7 interacts with the components of TFIIIC2 GTF3C1 and
GTF3C3 which is important for POLIII mediated gene transcription this
interaction was further confirmed by various in-vitro interaction studies in
which complex ofSIRT7 and TFIIIC2 components were further complexed by RNA
POLIII components confirming the fact that SIRT7 is important for RNA POLIII
mediated gene transcription and protein synthesis.
Association of SIRT7 with the genetic regions of tRNA and 5sRNA was also seen
and SIRT7 knockdown resulted in reduction of tRNA levels clearly indicating
that SIRT7 is essential for RNA POLIII mediated gene transcription.

 

 

Post
transcriptional modification causes transcription halt during mitosis

As
we know during mitosis, rDNA transcription is halted and only cell division
occurs. After mitosis transcription resumes again with the help of SIRT7, SIRT7
undergoes conformational modification in the C-terminal before transcription
proceeds in late telophase11.During mitosis,SIRT7 is phosphorylated with CDK1-cyclin
B and dephosphorylated by a phosphatase sensitive to okadaic acid at the end of mitosis.This indicates that
dephosphorylated form is necessary for SIRT7 activation after mitosis to resume
transcription as this causes conformational modification of SIRT7 C-terminal. This
hypothesis was tested by raising antibodies against N-terminal region of SIRT7
but this did not show any variation in the level of SIRT7 signal during early
and late mitosis instead increase in reactivity was seen at C-terminal of SIRT7
with anti-C terminal antibody during late mitosis but this reactivity was low
during early mitosis clearly indicating that conformational change caused at
C-terminal region of SIRT7 by dephosphorylation in late mitosis is the cause
and very important for transcription restart Similarly, all other RNA
Polymerase1 component are phosphorylated and dephosphorylated before and after
mitosis respectively. For example, SL1/TIF1-IB is inactivated by cdc2-cyclin b
phosphorylation which does not allow its interaction with UBF to initiate
transcription.

 

 

 

Summary

In spite of weak
catalytic activity of SIRT7 enhanced studies revealed that it is deeply
involved in rDNA transcription starting from initiation through elongation to
nascent rRNA processing. Its association with chromatin from the start of
transcription until processing indicates that it has a vital role in
transcription of rDNA and its also fascinating to know that it is bound to
chromatin even during mitosis rather being in NOR region as transcription gets
halted at that time.This area needs further research to investigate the
functional role of SIRT7 during mitosis.Although various studies reveal that it
is regulating rDNA transcription from initiation to processing but its
functional role needs to be validated by further techniques which will make its
role more clear and it is also needed to find out whether there is any other
deacetylating substrates in addition to PAF53 involved in rDNA transcription to
better understand the whole process.