Schizothorax richardsonii dsRNA dependent protein kinase has three double-stranded RNA binding motifs with a promoter lacking conserved kinase sequence

Background: Double stranded RNA (dsRNA) dependent protein kinase (PKR) is an interferon (IFN) stimulated antiviral protein. It inhibits protein synthesis by phosphorylation of the eukaryotic translation initiation factor 2-alpha (eIF2-α) by its serine-threonine kinase activity that prevents virus replication. This is the first report on the in silico analysis of PKR coding region and its promoter from a Coldwater fish of the Indian Himalayan region. Snow trout, Schizothorax richardsonii is an important Coldwater food fish. It is being over fished and therefore requires conservation. Being a vulnerable species it is listed under the red list of IUCN. Here we discuss the identification, cloning and sequencing of PKR coding region and its promoter. Results: We have revealed the complete coding region of dsRNA dependent protein kinase (PKR) and its promoter from Schizothorax richardsonii , one of the several species of snow trout inhabiting sub-Himalayan fresh water bodies. An amplicon of 2884bp containing 5ʹ and 3ʹ untranslated regions (UTR) of 234 and 558 bases was obtained while the deduced open reading frame (ORF) of 2076 bases encoded a polypeptide of 691 amino-acids. Snow trout PKR protein contains three double stranded RNA binding motifs (dsRBM) at N terminal, besides possessing a serine/threonine protein kinase and a C terminal catalytic domain. Moreover, a stretch of 791 nucleotide bases was identified as the promoter upstream the ORF. The identified promoter has two interferon stimulated response elements (ISRE) besides the presence of core promoter elements. Moreover, the snow trout PKR promoter has a TATA box but lacks kinase conserved sequence (KCS) that is present in mammalian PKR promoters. Conclusion: The promoter of snow trout was identified as a stretch of 791 nucleotide bases. It has two interferon-stimulated response elements (ISRE) besides core promoter elements. Intriguingly, unlike the mammalian PKR promoter, the snow trout PKR promoter has a TATA box but lacks the conserved kinase sequence (KCS) present in human counterpart. Double stranded RNA; dsRBM- RNA binding motifs; DPE- Downstream promoter element; eIF2-α- eukaryotic translation initiation factor 2-alpha; EXPASY- Expert Protein Analysis System; IFN- Interferon; ISRE- Interferon stimulated response elements; ISG- Interferon stimulated gene; KCS- Kinase conserved sequence; NLS-Nuclear localization signal; N-Lobe- N-terminal lobes; ORF- Open reading frame; PKR- Protein kinase; pI- Isoelectric point; PSI-BLAST- Position-specific iterated-Basic Local Alignment Search Tool; Sr- Schizothorax richardsonii; TBE- Tris Borate EDTA; TSS- Transcription start site; UTR- Untranslated regions

The identified promoter has two interferon stimulated response elements (ISRE) besides the presence of core promoter elements. Moreover, the snow trout PKR promoter has a TATA box but lacks kinase conserved sequence (KCS) that is present in mammalian PKR promoters.
Conclusion: The promoter of snow trout was identified as a stretch of 791 nucleotide bases. It has two interferon-stimulated response elements (ISRE) besides core promoter elements. Intriguingly, unlike the mammalian PKR promoter, the snow trout PKR promoter has a TATA box but lacks the conserved kinase sequence (KCS) present in human counterpart. Keywords: Schizothorax richardsonii , Interferon Stimulated Genes, PKR protein, Antiviral state, Homology modeling, dsRNA binding protein kinase Background The protein kinase R (PKR) is an interferon-inducible dsRNA dependent protein kinase with antiviral properties. It is known to limit viral replication by blocking eIF2α mediated translation. PKR is a Serine/Threonine kinase that phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF-2α) which in turn leads to the inhibition of protein synthesis [1]. PKR is known to be expressed constitutively at moderate levels in cells besides being activated by viral dsRNA as well as ds RNA analog poly I:C. Viral infection leads to induction of interferon and PKR is induced as an effector protein against viral invasion [2].Besides the antiviral activity, PKR protein also responds to various cellular stresses, regulates cell growth and also has a role in inflammatory processes, apoptosis, and metabolism [3]. PKR protein is composed of two major domains, one dsRNA binding domain (dsRBD) and a kinase domain. The dsRBD consists of two to three tandem dsRNA binding motifs (dsRBM) [3]. Virus-derived dsRNA binds to the dsRBD enabling homo-dimerization and auto-phosphorylation in a stretch of amino-acids known as activation segment.
In the activation segment, residues Thr 446 and Thr 451 are steadily phosphorylated during the process of activation. Active PKR is known to bind and phosphorylate Ser 51 residue of eIF-2α that results in the activation of the kinase domain [4,5].
PKR has been identified from a number of fish like grass carp, rock beam, Japanese halibut and fresh water pufferfish [6,7,8,9]. In puffer fish Tetraodon nigroviridis, three PKR genes are known to be induced by poly I:C, one containing three doublestranded RNA binding domain (dsRBD) and two containing one dsRBD domain. On the contrary in zebra fish, one PKR has been described with three dsRBD. In Fugu, three-spined stickle back and fat head minnow two PKR genes with a putative dsRBD have been identified. Whereas one gene with two dsRBD has been reported in Japanese media. In Japanese flounder one, PKR gene with similar characteristic features like that of mammalian PKR has been described. It has been demonstrated that over expression of PKR increases the phosphorylation of eIF-2α and inhibits the replication of turbot (Scophtalmus maximus) rhabdovirus in embryonic cells [8].
Functional analysis of dsRNA binding motifs has been carried out in grass carp where the third dsRBM has been shown to enhance dsRNA binding [10]. Thus, the knowledge about fish PKR is quite intriguing which needs to be further unravelled and application of bioinformatic tools can prove helpful in this direction. The knowledge on three dimensional structures of fish PKR is inadequate and a comparative structural and sequence analysis of the fish PKR with its mammalian orthologs thus needs in-depth exploration. This will help in understanding the fish immune mechanisms and will generate knowledge about different ways the immune system adopts to combat the pathogen attack. We have attempted to identify and characterize PKR and its promoter from an Indian Coldwater fish, snow trout.
Schizothorax richardsonii one of the several species of snow trout, is an important conserved cyprinid fish of the mountainous riverine system. It is an economically important food and game fish facing a serious risk. Overfishing, pollution of aquatic bodies, damming of rivers and introduction of salmonids and other exotics have threatened its existence. Therefore, it has been listed as vulnerable species as per the IUCN red list of threatened species (2010). In order to identify and characterize PKR and its promoter from a Coldwater fish species inhabiting the foothills of Himalayas, we have identified, cloned and sequenced the PKR coding region and its promoter. Moreover, this is first report on the in silico analysis of PKR coding region and its promoter from a Coldwater fish, Schizothorax richardsonii.

Amplification of PKR gene
The nucleotide sequence obtained by PCR was analyzed, and partial coding region of dsRNA dependent protein kinase of 900bp was submitted to GenBank (Accession No. KX447496). Two fragments of 1400bp and 800bp were obtained by 5' and 3' RACE analyzed on 1% agarose gel documented using GelDoc XR (BioRad), after sequencing and analysis of RACE PCR products, a full-length PKR nucleotide sequence of 2884 bases were obtained. Bioinformatic analysis of the obtained sequence revealed that it has an open reading frame (ORF) of 2076 nucleotides with a 5ʹ UTR of 234 nucleotides and a 3ʹ UTR of 558 nucleotides. The complete coding sequence was submitted to GenBank (Accession No. KX447496.1). 3ʹ UTR was predicted to have four instability motifs (ATTTA) and one polyadenylation signal (AATAAA) before PolyA tail (Fig 1).

Amplification of PKR promoter region
To identify the PKR promoter region, genome walking was carried out. PCR products were obtained from the genomic libraries acquired by the complete digestion of

Analysis of PKR Protein
The snow trout PKR protein was predicted to be a polypeptide of 691 amino acids.
The molecular weight of the protein was predicted to be 77.954 kDa with a predicted isoelectric point (pI) as 8.71. Domain analysis revealed the presence of two major domains a dsRNA binding domain and a Ser/Thr kinase domain (Fig 3).
The dsRNA binding domain has three dsRNA binding motifs (dsRBM) while an ATP binding site "IGKGGFGRVFKARRKLEKKYFAVKI" between position 408-431 and a kinase active site "LIHRDLKPKNIMF" between positions 541-553 in the kinase domain. PKR also possesses a bipartite nuclear localization signal and four N-Glycosylation sites in the dsRBD.

Homology Modelling
The snow trout dsRBM1 has 41% identity with a first dsRBM domain of mouse PKR (PDB ID: 1x49 A) while dsRBM2 and dsRBM3 had 30% and 44% identity with a second dsRBM domain of mouse PKR (PDB ID: 1x49 A). The kinase domain had 44% identity with human PKR kinase domain (PDB ID: 3UIU). Therefore it was selected as a template to develop a homology model. The dsRBM1 and dsRBM3 had a typical feature of mammalian dsRBMs as it has three β-sheets sandwiched between two αhelices (Fig 4). However, the dsRBM2 differs from the other two dsRBMs as it has two β-sheets sandwiched between two α-helices (Fig 4) whereas PKR kinase domain is formed of β-sheets at its N-terminal and α-helices at C-terminal ( Fig 5). The Nlobe of the kinase domain consists of 5 stranded antiparallel β-sheets and one αhelix, which contains the ATP binding site. The C-lobe of the kinase domain consists of eight α-helices and two pairs of antiparallel β-sheets.

Phylogenetic analysis
Evolutionary analysis of PKR protein from various fish species was carried out using maximum likelihood method in MEGA7 to get a phylogenetic tree along with PKR from Schizothorax richardsonii (Fig 6). PKR of fish and mammalian species separated into two major clades one including the fish while the other includes the mammalian species. One of the fish species Larimichthys crocea, of Sciaenidae family separated itself from fish as well as mammalian species, forming a separate clade quite distant from both. PKR of Schizothorax richardsonii grouped itself among other members of Cyprinidae family. Moreover, the evolutionary analysis of fish and human RNA binding motifs (dsRBMs) revealed that the dsRBM from fish and human clustered into three separate clades. The first clade includes the dsRBM1 of fish along with dsRBM2 of a human while dsRBM2 of fish grouped itself with dsRBM1 of human (Fig 7). However, the third dsRBM of fish (dsRBM3) clustered itself into a separate clade. The fish species included are Schizothorax richardsonii (Sr), Carassius auratus (Ca) and Ctenopharyngodon idella (Ci).

Discussion
Initially, a partial sequence of snow trout PKR was amplified, and the complete sequence was deciphered by 5′ and 3′ RACE using the primers derived from the  [1,11]. Nucleotide sequencing of the amplified promoter region of PKR gene revealed the presence of two ISREs. PKR promoter has the characteristic features of a core promoter as it has a transcription start site (TSS), a downstream promoter element (DPE), a TATA box beside two ISREs. Intriguingly, human and mouse PKR promoter regions are without TATA box and contain a unique kinase conserved sequence (KCS) which is a 15bp sequence [12]. However, KCS is absent in the PKR promoter of snow trout. KCS is a unique feature of TATA-less PKR promoter and responsible for the increased ISRE activity of the promoter. It has a role in both optimal basal expression and interferon-induced expression of PKR protein [11]. So, the absence of KCS in snow trout PKR promoter can be implicated to the presence of TATA box within its promoter which is a prerequisite for the basal expression and IFN induced expression of the gene.
Snow trout PKR coding sequence encodes a polypeptide of 691 amino acids with a molecular weight of 77.954 kDa and isoelectric point (pI) 8.71. The protein contains a bipartite nuclear localization signal (NLS) affirming that the snow trout PKR is a nuclear protein. In humans, PKR has been demonstrated to have both nuclear and cytoplasmic localization [13]. However, the bipartite nuclear localization signal in snow trout PKR suggesting its translocation into the nucleus. The role of the cytoplasmic form of PKR is well known to inhibit translation initiation factor, eIF2-α while the role of the nuclear form of PKR is not very evident. However, the role of the bipartite nuclear localization signal in nuclear signalling has been suggested [14]. It will be quite interesting to know whether knocking out the bipartite NLS of snow trout PKR retains its function besides localization in the cytoplasm.
The PKR protein possesses two domains, a dsRNA binding domain (dsRBD) and a kinase within it. The dsRBD consists of dsRNA binding motifs (dsRBMs) that help to bind the dsRNA while, the catalytic domain or the kinase domain is a serine/threonine kinase [3,6]. So far, a complete homology model for PKR has not been reported, however, separate models for both PKR kinase domain and dsRNA binding domain (dsRBD) have been described [1,6,9]. Here we have attempted to predict the 3D structure of the dsRNA domain and kinase domains using homology modelling. Snow trout PKR has three dsRNA binding motifs (dsRBMs) within the dsRBD, while the reference mouse model had only two. So, individual models were developed for each dsRBM and kinase domain. Snow trout dsRBM1, dsRBM2, and dsRBM3 were having a maximum identity with the first dsRBM of mouse PKR (PDB ID: 1x49A) thus used as a template for the homology modelling of the three motifs.
The dsRNA binding domain (dsRBD) consists of two or three dsRNA binding motifs (dsRBM) [6]. dsRBD of snow trout PKR has three tandem dsRBMs. Presence of three tandem dsRBMs in dsRNA binding domain has also been reported from PKRs of other fish [6,9]. Two dsRBMs are reported from mammalian PKR while three tandem dsRBMs has been reported universally in fish [6,9]. Three PKR genes are known to be induced by poly I:C in puffer fish one containing three double-stranded RNA binding domain (dsRBD) while two possess one dsRBD domain. However, one PKR has been described with three dsRBD in zebra fish. Two PKR genes with a putative dsRBD have been identified in fugu, three-spined stickle back and fat head minnow.
Whereas Japanese media has one gene with two dsRBD, but the lone PKR gene of Japanese flounder has similar characteristic features like that of mammalian PKR [8]. The dsRBM1 of fish is closely related to dsRBM2 of a human while dsRBM2 of fish is more closely related to dsRBM1 of human, while the third dsRBM3 of fish is unique and closely related across fish species. The dsRBM1 and dsRBM3 had a typical feature of mammalian dsRBMs that is three β-sheets sandwiched between two α-helices. The dsRBM2 varies from the other two dsRBMs in the number of βsheets as it has only two β-sheets sandwiched between two α-helices. However, the evolutionary analysis suggests that dsRBM1 and dsRBM2 are related to the two dsRBMs of human PKR while dsRBM3 is exclusive to fish and hence a unique feature to certain fish. Functional analysis of the three dsRBMs from grass carp revealed that the dsRBM1 and dsRBM2 are essential for the function of PKR while the third dsRBM3 enhances the function of PKR [10].
PKR kinase domain has the Serine/Threonine kinase activity that works either by auto-phosphorylation or phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) [1]. The kinase domain has an ATP binding site and a kinase active site. At the N-terminal of the kinase domain of PKR, an ATP binding site is present that contains a conserved glycine-rich stretch close to a lysine residue [15]. The The PKR kinase domain is formed of two lobes-an N-terminal lobes (N-Lobe) formed of β-sheets while the other C-terminal lobe (C-Lobe), formed of α-helices [17]. Nlobe of the kinase domain in human is formed of five antiparallel β-sheets and two α-helixs. One helix is at the top and the other one at the base whereas the helix at the base contains ATP binding site [17]. The N-Lobe of snow trout PKR kinase domain contains five antiparallel β-sheets and one helix at the base but lacks a helix at the top. ATP binding site or the functional site of the N-lobe is present in the α-helix at the base indicating that the snow trout PKR kinase domain retains the functional site. Furthermore, the C-lobe of human PKR kinase domain is composed of eight α-helices and two pairs of antiparallel β-sheets [17]. The C-Lobe of snow trout PKR kinase domain also contains the two pairs of antiparallel β-sheets with six αhelices. Although the kinase domain in snow trout lacks some structural features of human PKR kinase domain, but it retains the essential functional features of the PKR kinase domain.
The PKR protein of snow trout is a nuclear form of dsRNA-dependent protein kinase.

Cloning and sequence analysis of PKR gene
Fish acclimatized to laboratory conditions for a week were injected intraperitoneally with poly (I:C) at a dose of 200µg per fish in a volume of 25µl [18] using a tuberculin syringe. Prior to dissection, the fish were anesthetized by dipping them in 0.05ml/l clove oil for 15-20 min [19]. Liver, spleen, and kidney were collected from treated fish after 72 hours of treatment. Total RNA was isolated from the collected tissues using Ribozol (Amresco) according to manufacturer's recommendations.
cDNA was prepared using total RNA as template and reverse transcriptase (RevertAid Reverse Transcriptase, Thermo) in a reaction volume of 20µl containing 5µg total RNA, 0.5µg random hexamers, 20pmol dNTP mix, 20U RNase inhibitor and 200U of RevertAid Reverse Transcriptase. The reaction was incubated at 25ºC for 10min followed by 42ºC for an hour. Finally, the contents were heated at 70ºC for 10 min to stop the reaction. Primers were designed from the conserved region PKR coding sequences of Cyprinids after aligning the available sequences (Table 1). A fragment of approximately 900 base pairs was amplified, in a reaction volume of 25µl using 2.5µl of standard Taq Buffer, 10pmol of forward and reverse primers, 10pmol of dNTP mix, 1.5mM MgCl2, 1.25U of Taq DNA polymerase (NEB) and 2.5µl of cDNA template. PCR reactions were amplified by initial denaturation at 95ºC for 5min, followed by 35 cycles of denaturation at 95ºC for 45s, primer annealing at 60ºC for 1min, primer extension at 72ºC for 1min and a final extension of 10min at 72ºC. A PCR product of expected size (900 base pairs) was gel purified, cloned using InsTAclone PCR cloning kit (Thermo Scientific) and outsourced for nucleotide sequencing.

Rapid Amplification of cDNA Ends -Polymerase chain reaction (RACE-PCR)
Gene-specific primers for 5' and 3' RACE were designed (Table 2) using the partial nucleotide sequence of snow trout PKR coding region (Accession No. KX447496).
Using SMARTer® RACE 5'/3' Kit (Clontech) 5' and 3' ends of the PKR gene were amplified following the manufacturer's recommendations. Cycling conditions included 35 cycles of denaturation at 94ºC for 30s, annealing at 68ºC for 1min and primer extension at 72ºC for 3 min. The two fragments 1400bp (5' RACE) and 800bp (3' RACE) obtained were cloned using InsTAclone PCR cloning kit (Thermo Scientific) and outsourced for nucleotide sequencing.

Nucleotide Sequence Analysis
The 3ʹ UTR was analyzed while 5 ʹ UTR was analyzed for the presence of any regulating elements within it using YAPP (http://www.bioinformatics.org/yapp/cgibin/yapp.cgi) and JASPER [20].

Amplification and cloning of PKR promoter region
Genome walking was performed to identify the PKR promoter region. Briefly, genomic DNA was isolated from snow trout liver and kidney using Wizard® Genomic with universal adaptor were used as template for primary PCR along with forward primer (AP1) a universal adaptor primer 1, along with a reverse primer (PKRproGSP-1) as gene-specific outer primer (Table 1). Secondary PCR was carried out using primary PCR product as template along with universal adaptor primer 2 (AP2) as forward primer and gene-specific inner primer (PKRproGSP-2) as reverse primer ( and JASPER [20] were employed.

PKR protein analysis
The coding sequence of PKR was translated by Expert Protein Analysis System (EXPASY) translation tool [21]. Deduced protein sequence was analyzed for various structural and functional features using online prediction tools. The physicochemical properties of the protein were unravelled using ProtParam [22]. Domain analysis of the protein was carried out to reveal the functional features of the PKR protein. The protein domain features were predicted using PROSITE [23]. To interpret the Nglycosylation sites in the protein, NetNGlyc was used (http://www.cbs.dtu.dk/services/NetNGlyc/). The sub-cellular localization of the protein within the cell was predicted using WoLF PSORT II and the nuclear localization signal was detected by cNLS mapper [24][25].

Phylogenetic analysis
To carry out phylogenetic analysis of PKR protein from various fish and mammalian species, PKR protein sequences were retrieved from GenBank. The protein sequences were aligned using ClustalW in MEGA7 software [26]. Evolutionary history was inferred by using the maximum likelihood method based on the JTT matrixbased model [27]. Maximum parsimony method was applied for automatic heuristic search to obtain initial trees. Tree was drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 [26].
Moreover, the dsRNA binding motifs of fish Schizothorax richardsonii (Sr), Carassius auratus (Ca) and Ctenopharyngodon idella (Ci) were aligned using ClustalW along with two dsRNA binding motifs of human PKR protein for analyzing the evolutionary relationship.

Homology Modelling
To develop the homology model for PKR protein, a template model was required. To obtain such template, position-specific iterated BLAST (PSI-BLAST) was carried out.
Stereo-chemical quality of the models was validated by constructing Ramachandran Plot using PROCHECK [12]. Moreover the accuracy of the models was also validated in Verify_3D using SAVES (services.mbi.ucla.edu/SAVES/). Ethics approval and consent to participate All applicable international, national, and/or institutional guidelines (Institutional Animal Ethics Committee of ICAR-DCFR) were followed for the care and use of animals during the study.

Consent for publication
Not Applicable

Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interest
The authors declare that they have no competing interest.

ARRIVE Guidelines
Not applicable as no in vivo studies were carried out.  Phylogenetic analysis of PKR with Maximum likelihood method in MEGA7.

Figure 7
Evolutionary analysis of dsRNA binding motifs (dsRBM) of fish and human. The fish species in