Abstract SIRT7 is involved in regulating both POLI-

AbstractLocalization of Sirt7in nucleolus indicated that it is involved in rDNA transcription and ribosomebiogenesis. 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 aresubsequently transcribed by RNA polymeraseI.

SIRT7 was found to be associatingwith RNA POLI for rDNA transcription. Studies also showed that SIRT7 interactswith various components of TFIII2 complex and components of RNA POLIII complex involvedin tRNA and 5sRNA gene transcription, although, SIRT7 overexpression did notincrease protein synthesis indicating its indirect involvement in proteinsynthesis. The ribosomal protein synthesis gets affected by Sirt7 and RNA POLIIIassociation and dissociation. Earlier study results clearly suggest that SIRT7is involved in regulating both POLI- and POLIII-dependent transcription.

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Herewe present a detailed review report of SIRT7 involvement in rDNA transcriptionand protein synthesis and its role in cellular stress. IntroductionEpigenetics has now become the most studied area asit is seen to be involved extensively in gene regulation. It is widely acceptedby the living system because of its ability to be reversed and can betransferred to next generation. Epigenetics constitutes post translationalmodification as the main machinery for regulating gene expression among whichDNA methylation, chromatin remodeling and histone acetylations /deacetylationsare some of the extensively used mechanisms, histones are the major target ofthese processes for both repressing and activating a particular gene to carryout a molecular process. Histone deacetylases (HDAC’s) are the enzyme whichcarry out deacetylations of acetylated histones by removing acetyl groups froman?-N-acetyl lysine amino acid on a histone making it more compact and unavailable forany molecular process. Sub-families of HDAC are class1-class4 and here we willconcentrate on a class3 HDAC called sirtuins7. Unlike all other HDAC’s sirtuinshas a  NAD+ binding catalytic domain andwidely distributed in nucleus (SIRT1,2,6,7), cytoplasm (SIRT1,2), mitochondria(SIRT3,4,5).Amongst all the sirtuins sirt1 and sirt7 is involved in variousmetabolic and cellular functions like energy metabolism, DNA damage, regulatesfat oxidation in liver, ageing in lower vertebrates, in higher vertebratesinspite of directly involved in ageing regulation it is regarded as a metabolicfactor.

Its localization in nucleolus indicates its involvement in ribosomebiogenesis or protein synthesis and this has been proved by various researchersbecause ribosomes are found in close proximity with the nucleolar region. Sirt7 is located onchromosome 17 in subtelomeric region with 10 exons and an ORF of 1203bpencoding a protein length of 400 amino acids. It has a molecular weight of 44KDand its catalytic domain is present between 90 and 331 amino acid residues. Innormal conditions it is highly expressed in metabolically active tissues likeCD-33 positive myeloid bone marrow precursor cells, liver and almost alltissues except non proliferating tissues like muscle, brain and ovary. Like allother sirtuins Sirt7 has a weak deacetylase activity, some of the knownsubstrates are p53, h3k18ac, NPM1, PAF53,GABPbeta1.

 table1. ShowingInteracting partners and substrates of SIRT7 Our focus here isPAF53 substrate the deacetylase activity of SIRT7 is also involved in rDNAtranscription. RNA polymerase1 is responsible for rDNA transcription which isrequired for synthesis of ribosome. Metabolically active cells are in need ofcontinuous ribosome synthesis and function for which Sirt7 deacetylates PAF53(at position k373) a subunit of RNA polymerase1 this activity of SIRT7 isrequired for RNA pol1 binding to rDNA promoter for transcription initiation andelongation2. SIRT7 and rDNA transcription Ribosomal RNA issynthesized in nucleolus and the genes for this RNA is clustered in fivedifferent chromosomal tips found in nucleolus3.

SIRT7is found to be closely associated with rDNA promoter along with RNA polymeras1and this was proved when RNase treatment caused redistribution of Sirt7 fromnucleoli to nucleoplasm indicating association of SIRT7 with RNA plymerase1during transcription in nucleolus. Co-immunoprecipitation assays involvingantibodies against RNA polymerase1 but not control antibodies co-precipitatedendogenous SIRT712.Similarly, when RNA Polymerase1 wasco-precipitated with alpha FLAG(M2) antibody against FLAG tagged SIRT7 clearlyindicating that SIRT7 interacts with RNA polymerase1 machinery to carry outtranscription12. Knockdown of SIRT7 caused significantdecrease in rRNA synthesis followed by lesser association of RNA polymerase1 tothe rDNA promoter and overexpression caused increase in RNA ploymerase1mediated transcription4. This clearly indicates functional role ofSIRT7 in rDNA transcription but clear functional characterization of SIRT7 isnot specified.

As we know histonedeacetylase do not have direct DNA binding domain so they need an additionalprotein with a DNA binding domain. Hence, It has been reported thatUBF(upstream binding factor) which binds to the upstream region of rRNA genesand recruits other factors required for forming pre-initiation complex likeSL1, SL1 consists of TATA binding protein(TBP) and TAF’s.This association wasmore clear when sertinol an inhibitor of sirtuins caused inhibition of rDNAtranscription.5.It is reported that SIRT7 is foundassociated with UBF in most cell cycle stages(G1, G1/S, Sphase) and even duringmitosis SIRT7 and UBF are complexed and remain bound to chromatin instead ofresiding in NOR’s.As discussed aboveSIRT7 has a direct deacetylating substrate PAF53 which is a component of RNApolymerase1 complex this association of SIRT7 and PAF53 is essential forrecruitment of RNA polymerase1 on rDNA promoter by interacting with othersubunits and UBF6. Co-immunoprecipitation experimentsconfirmed the association of SIRT7 and PAF53.

The association of PAF53 with RNAPOLI was not affected by both wild type and mutated PAF53(K373) suggesting thatacetylation does not affect interaction of PAF53 with POLI.MutatedPAF53(arginine in place of lysine) revealed high pre-rRNA synthesis indicatinghypoacetylation is important for rRNA synthesis6.Duringstress, when SIRT7 is released from nucleoli the association of PAF53 withSIRT7 and subsequently with RNA polymerase1 is lost due to hyperacetylation ofPAF53 and thereby halting rDNA transcription6.Pre-rRNAsynthesis was reduced in both cases when CBP and PAF53 was knocked downseparately suggesting that both acetylation and deacetylation by SIRT7 isnecessary for rRNA synthesis but PAF53 occupancy was slightly increased in CBPdepleted cells.During stresseslike glucose deprivation or due to some treatment when SIRt7 is released fromnucleoli rDNa transcription is halted and hyperacetylation of PAF53 is seenwhereas in mutated PAF53 (K373R) no acetylation was observed indicating thatK373 is the only lysine residue which is deacetylated during stress.

Perturbedinteraction between PAF53 and SIRT7 was seen during stress which led tohyperacetylation of PAF53 but the association of PAF53 with RNA POLI wasaffected only a little by 30% but not to a great extent contradicting the factthat deacetylated PAF53 is required for RNA POLI and rDNA association.Treatmentof cells with RNAse A showed that binding of SIRT7 and PAF53 depends upon RNAbecause SIRT7 and PAF53 interaction was abolished.On the other hand this RNAseA treatment did not affect UBF and rDNA interaction whereas SIRT7 was releasedfrom nucleoli indicating that SIRT7 protein-protein interaction and itsretention in nucleoli is dependent on RNA.Another interactingpartner of SIRT7 involved in rDNA transcription is RPA194(table1) the largestsubunit of RNA polymerase1.

Anti-RPA194 affinity resin incubation withtranscriptionally competent cell extract showed failure in transcribing rRNAgenes. This subunit is involved in elongation process of transcription as ithelps in movement of RNA POLI enzyme complex three bases ahead duringelongation this characteristic feature became more clear when RPA194, RPA127,RPA40 and PAF53 eluted in the same immunoblots but UBF(involved intranscription initiation) co-elution was not found7.Although functional characterization of RPA194 is still not done butknockdown of SIRT7 by siRNA in hela, A549, U2OS,ZR75 cells showed significantdecrease in the protein level of RPA194 but deacetylation activity of SIRT7 onRPA194 is yet not stated8.Co-localisation of SIRT7 and RPA194 was nothampered by RNAse treatment suggesting SIRT7 and RNA POLI participate in tightinteraction.Sirt7and chromatin remodeling complexesAccording to theproteomic analysis data of Tsai et all SIRT7 is found to interact with thecomponents of B-WICH chromatin remodeling complexes like WSTF, Mybbp1A, SNF2H,etc.

According to the STRING database results all the components of B-WICH andother chromatin remodeling complexes are found to be associated with SIRT7 butthe direct interactions and functional link between these proteins were stillmissing. To get more clear picture of SIRT7 functionality the  number of associated partners were  narrowed down by checking the abundance ofthe protein in immunoisolations alongwith SIRT7 and calculating the NSAF valueswhich were further normalized by PAX database14.Theresults showed that the components of B-WICH were highly abundant for exampleSMARCA2, SMARCA4, RSF1, BAZ1B, BAZ2A, TTF1 these are the proteins involved inremodeling of the chromatin regions involved in rRNA synthesis.

Since this datadoes not provide the clear picture of SIRT7 involvement in chromatin remodelingby direct interaction with the components it could be associated with B-WICHvia some other protein which needs to be addressed. SIRT7 and protein synthesisSIRT7like yeast Sir2 is localised in nucleoli which is the center for ribosomebiogenesis SIRT7 is involved in protein synthesis also as its knockdown inliver cancer cells showed decrease in the level of ribosomal proteins9.SIRT7 was found to be associated withTFIIIC2 components in affinity purification and mass spectrometry analysis.WhenSIRT7 was found interacting with RNA polymerase III components (POLR3B, POLR1D,POLR3A, POLR1C, POLR3C, POLR3D,POLR2H and POLR2E) and TFIIIC2 complex (GTF3C1, GTF3C2, GTF3C3, GTF3C4, GTF3C5and GTF3C6) 8.Sinceribosome are the translational machinery and RNA polymerase III is involved intRNA and 5srRNA SIRT7 knockdown loses its association with the above mentionedtranslational components thereby halting translation of ribosomal proteins. Onthe contrary, SIRT7 overexpression did not increase ribosomal protein synthesisindicating an indirect effect of it on translation but SIRT7 overexpressioncaused decrease in the level of ribosomal protein RPS7 and RPS14, this may bemediated though MYC regulated SIRT7 association to ribosomal protein geneticregions to decrease ER stress 10.

Knockdown of SIRT7 caused decrease in both proteinsynthesis and rDNA transcription but inhibition of protein synthesis waspreferred over rDNA transcription.8. In order todetermine the involvement of SIRT7 in protein synthesis interaction betweenSIRT7 and RPL11 a protein found in 60S subunit was investigated and the resultshowed that SIRT7 and RPL11 show co-localisation in monoribosomal regions andare palying an important role in cell cycle progression.SIRT7 knockdownresulted in release of RPL11 to polyribosome region and subsequent silencing ofSIRT7 by siRNA and to study its effect in protein synthesis by measuring the levelof amino acid analog HPG incorporation showed that SIRT7 is necessary forprotein synthesis.Although SIRT7knockdown preferentially inhibits protein synthesis over rDNA transcriptionSIRT7 overexpression did not increase protein synthesis significantlyindicating that SIRT7 is involved in regulating protein synthesis via variouspathways and not through a single pathway.SIRT7 regulatesprotein synthesis by interacting with mTOR and TFIIIC2 forming a complex andaffecting the function of RNA POLIII.mTOR is a nucleolar protein and its localizationthere alongwith SIRT7 suggests its efficient involvement in proteinsynthesis.After all these studies and confirmation about SIRT7 involvement inprotein synthesis and ribosomal biogenesis it was essential to study itsdeacetylation activity on the interacting partners for which v global acetylationlevels of nucleolar proteins were measured before and after SIRT7 knockdown andthe results showed that there was not much effect on the levels of acetylationglobally before and after treatment however SIRT7 had protein and tissuespecific deacetyalase activity which may be responsible for its unique functionin regulation of rDNA transcription and protein synthesis.

Immunoaffinitystudies revealed that SIRT7 interacts with the components of TFIIIC2 GTF3C1 andGTF3C3 which is important for POLIII mediated gene transcription thisinteraction was further confirmed by various in-vitro interaction studies inwhich complex ofSIRT7 and TFIIIC2 components were further complexed by RNAPOLIII components confirming the fact that SIRT7 is important for RNA POLIIImediated gene transcription and protein synthesis.Association of SIRT7 with the genetic regions of tRNA and 5sRNA was also seenand SIRT7 knockdown resulted in reduction of tRNA levels clearly indicatingthat SIRT7 is essential for RNA POLIII mediated gene transcription.  Posttranscriptional modification causes transcription halt during mitosisAswe know during mitosis, rDNA transcription is halted and only cell divisionoccurs. After mitosis transcription resumes again with the help of SIRT7, SIRT7undergoes conformational modification in the C-terminal before transcriptionproceeds in late telophase11.

During mitosis,SIRT7 is phosphorylated with CDK1-cyclinB and dephosphorylated by a phosphatase sensitive to okadaic acid at the end of mitosis.This indicates thatdephosphorylated form is necessary for SIRT7 activation after mitosis to resumetranscription as this causes conformational modification of SIRT7 C-terminal. Thishypothesis was tested by raising antibodies against N-terminal region of SIRT7but this did not show any variation in the level of SIRT7 signal during earlyand late mitosis instead increase in reactivity was seen at C-terminal of SIRT7with anti-C terminal antibody during late mitosis but this reactivity was lowduring early mitosis clearly indicating that conformational change caused atC-terminal region of SIRT7 by dephosphorylation in late mitosis is the causeand very important for transcription restart Similarly, all other RNAPolymerase1 component are phosphorylated and dephosphorylated before and aftermitosis respectively. For example, SL1/TIF1-IB is inactivated by cdc2-cyclin bphosphorylation which does not allow its interaction with UBF to initiatetranscription.   Summary In spite of weakcatalytic activity of SIRT7 enhanced studies revealed that it is deeplyinvolved in rDNA transcription starting from initiation through elongation tonascent rRNA processing. Its association with chromatin from the start oftranscription until processing indicates that it has a vital role intranscription of rDNA and its also fascinating to know that it is bound tochromatin even during mitosis rather being in NOR region as transcription getshalted at that time.

This area needs further research to investigate thefunctional role of SIRT7 during mitosis.Although various studies reveal that itis regulating rDNA transcription from initiation to processing but itsfunctional role needs to be validated by further techniques which will make itsrole more clear and it is also needed to find out whether there is any otherdeacetylating substrates in addition to PAF53 involved in rDNA transcription tobetter understand the whole process.