DBHS Proteins

Discuss about the Relationship between Xenopus Tropicallis and Human SFPQ.

The aim of this literature review is to probe the interaction between DBHS and RNA. Specifically, the literature review focuses on the exploration of relationship between Xenopus tropicalis and the splicing factor proline or glutamine-rich protein (SFPQ). Xenopus tropicalis is swiftly being embraced as a model organism for developmental biology study and has enormous potential for raising the understanding of how the development of the embryo is controlled. A well-organized program with Xenopus community funded by National Health support in the United States, to enable the development of X. tropicalis as a novel genetic model system with the capability of influencing diver research field. The Xenopus has extensively been utilized in the past in studying various cell and developmental biology aspects. It offers the pros of a non-mammalian system which encompass high fecundity, external developmental as well as simple housing needs with extra benefits of enormous embryos, extremely conserved developmental courses, alongside close evolutionary connections to higher vertebrates. The Xenopus tropicalis remains an effective model in the cell biological studies.    

The DBHS proteins definition is through an extremely preserved tandem called N-terminal RRMs. This is a NonA/paraspeckles domain, NOPS alongside a C-terminal coiled-coil as seen in the figure 1 below:

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872119/

 To the exterior of the above conserved area, the family members differ substantially, both in sequence complexity and length as indicated above. The family has been enlarged as well as diversified to generate multiple paralogs purely within the invertebrates and vertebrates.

The DBHS are both nucleic acid alongside protein-binding dimers that can form order oligomeric complex as seen in the figure below as observed in figure 1 above. The DBHS proteins, in their structured core, remains highly modular, possessing protein-protein alongside protein-nucleic acid binding sites which allow them to act as a “molecular scaffold” as observed in figure 2 below:

Figure 2:

Past the structured area, N-alongside C-terminal low-complexity areas accounts substantially to functional diversity witnessed for paralogs of DBHS. The DBHS protein-binding sites alongside post-translation modifications mapped to X-ray crystal SFP  Q structure (4WIJ) (Pejaver et al, 2014)). The structure demonstrates a putative NONO/SFPQ heterodimer with other N-and C-terminal unfeatured and low-complexity domains that are modelled as flexible chains at the respective termini of structure. The sites for interaction within the X-ray crystal structure remain colored; while those of dimerization are green with the coiled-coil oligomerization motif being yellow. The secondary oligomerization site is colored brown whereas the putative RNA-binding surface of RRM1 is light blue (Brown et al, 2012). The putative RNA-binding loop of RRM2 is dark blue. The purple region illustrates the structurally unfeatured DNA-binding domain of SFPQ. The reported sites of post-translational modification alongside respective amino acid number are mapped as colored circles to NONO and SFPQ (Aparicio, Baer, R. and Gautier, J., (2014). The red indicates phosphorylation; the orange indicates methylation; the teal is the citrullination whereas the purple is the SUMOylation and the pale green regions indicate ADP-ribosylation (Davis, Chen and Chen, 2014). The asterisk represent the methylation sites subjected to citrullination. 

DBHS Proteins and Tumor Oncogenes

Each member of DHBS protein family has a conserved core of ∼300 amino acids described as “DBHS region” as seen in figure 1A above, the DBHS region entails the tandem different RRMs or the protein interaction NOPS domain alongside coiled-coil domain (Udayakumar and Dynan, 2015). The tandem RRMs in the DBHS protein are unique from each other and a flexible 7-amino acid linker separates them. The RRM remains the most abundant and well-featured nucleic acid binding domains, found in 0.5 to 1.0 percent of human genes (Knott et al, 2015). The DBHS RRM1 is defines as canonical, with conserved aromatic residues with extra extend beta-turns within loop three and five, one of which illustrates high conservation and takes after a double-stranded DNA/RNA  recognition motif (Knott, Lee, Passon, Fox and Bond, 2015). The C-terminal of edge of DBHS area has the highest charged coiled-coil domain that facilitate dimerization alongside oligomerization (Mircsof et al, 2015). The coiled-coil dimerization domain is forming the unusual right-handed antiparallel coiled-coil. Whereas modular in the DBHS protein core domain architecture, it possess which are probably to be disordered intrinsically (Liu, Qian and Cao, 2016).

Like several regulators of gene, DBHS proteins is either tumor oncogenes or suppressors in many transformed settings.  In respect of suppressors, tumors of breast with NONO loss remain linked to substantially surged tumor size, due to increased proliferation, may be aligns to NONO as cell cycle regulator. Likewise, SFPQ release from DNA aspects, or SFPQ or PTBP2 tumor suppression disruption complex permits swift cell proliferation alongside mitigation. In comparison, NONO remains shown as an extremely expressed in instances of malignant-melanoma, -pleural mesothelioma, -breast cancer as well as neuroblastoma (Bruelle et al, 2011).

Surged NONO abundance relates to increased melanoma progression, breast tumors malignant progression as well as lipid metabolism dysregulation. It is probably that NONO association with pervasive active transcription remains used to change the expression of gene in such settings. For instance, propelling enhanced neuroblastoma alongside bad patient results; NONO enhances concurrent contact with long noncoding RNA 1ncUSMycN alongside N-Myc mRNA culminating in post-transcriptional up regulating powerful neuroblastoma oncoprotein N-Myc. The NONO and SFPQ have further been connected to the promotion of growth and invasion in colorectal cancers as well as prostate cancer progression via AR-mediated task. The SFPQ is documented as oncogene via fusion with TFE3 in the papillary renal cell carcinoma alongside neuroblastoma or even bonded with ALB1 in the acute lymphoblastic leukemia. It is feasible in the latter case that dimerization as well as oligomerization features of SFPQ become harnessed by kinase fusion thereby promoting constitutive oncogenic kinase activity. SFPQ, fascinatingly, was documented lately as redistributing to membrane of cell in malignant cell lines of the leukemia patients, nevertheless, the implications functionally of this remains unknown.

RNA Binding Proteins and DBHS Proteins

The RNA binding proteins are significant in several aspects of cellular functions. Individually, the RNA binding proteins are helpful in the regulation of gene expressions at a multifaceted level, a course that remains de-regulated in various illness (Knott et al. 2015). One RNA family binding proteins known as DBHS proteins remain of interest because they are regulating gene at both bases, transcriptional alongside post-transcriptional. Moreover, the three members of family of DBHS protein in mammals including domain of Non-POU entailing NONO. This is called SFPQ alongside paraspeckles protein constituent 1 (PSPC1), together with theIncRNA NEAT1 remain all engaged in the sub-nuclear structures formation referred to as paraspeckles (Bond and Fox, 2009). 

The paraspeckles include the RNA: protein complexes situated in mammalian cell nuclei, and remain unusual because their formation, as well as maintenance, depends on a particular interaction between the NEAT1 and paraspeckles protein. The paraspeckles remain believed to play an imperative role in regulating gene expression through the sub-nuclear sequestration of particular proteins amongst them, DBHS proteins, hence attenuating their respective function. Moreover, the paraspeckles remain engaged in binding as well as retaining particular RNAs in the nucleus as a way of post-transcriptional regulation. Currently, there is a lack of evidence or knowledge relating to the molecular interactions taking place within the paraspeckles between RNA binding proteins and the RNAs they are interacting with. Such interactions remain central to bot function and formation of paraspeckles, and the constituent proteins together with RNAs.

Studies have been conducted through the optimization and application of PAR-CLIP (Photoactivatable ribonucleotide enhanced crosslinking and immunoprecipitation) that have helped efficiently isolate the RNA bound by DBHS proteins SFPQ and NONO. The isolated RNA has been subsequently sequenced while a bioinformatics examination pipeline implemented with program PARAlyzer hence identifying, to single nucleotide resolution, the SFPQ and NONO binding sites in the RNA (Kowalska et al, 2012).

Such a study have subsequently identified the periodic trend of SFPQ and NONO binding sites along the NEAT1_v2, feasibly a display of the oligomerization of proteins alongside architectural RNA.  This has finally provided significant insights into the internal organization of the paraspeckles complex capable of being applied in understanding additional IncRNA: Protein complexes. Moreover, such work has unearthed SFPQ bind to various biologically suitable IncRNA, thus proving a new function for the SFPQ in the gene expression regulation. Feasibly, such proteins regulate the cellular function of respective target IncRNAs (Zhang and Walter, 2014). The study has further identified several protein-coding RNAs bound by SFPQ. The preference for such proteins in binding to long first introns have been determined, feasibly for stabilizing nascent RNAs while they are being made. Moreover, various SFPQ bound RNAs to encode important molecules in the progression of cancer alongside many encode paraspeckles proteins hence implicating SFPQ in substantial illnesses and stress responses pathways.

PAR-CLIP Technique and SFPQ and NONO Binding Sites

The cell nucleus, primarily in the eukaryotes (complex), remains extremely structured. Each chromosome occupies distinct territories, and particular protein alongside nucleic acids that remain supplemented in the structures of subnuclear like nucleoli, paraspeckles, and Cajal bodies alongside nuclear speckles (Mjelle et al, 2015). The nuclear organization is connected to genome maintenance and gene expression control and hence impact development, growth alongside cellular proliferation. Also, nuclear organization disruption is usually linked to illness states like loss of sub-nuclear promyelocytic leukemia bodies in the acute promyelocytic leukemia. In the current review, the discussion the composition, formation as well as functions of the paraspeckles which is amongst the latest sub-nuclear bodies identified is done alongside the RNA to demonstrate their associations and the role of paraspeckles in the control of gene expression based on tapping adenosine to inosine (A to I) hyperedited RNA within nucleus (Mateo et al, 2015). Current evidence indicates that such mechanism might be broadly utilized to coordinate the expression of within a wide range of cellular settings. The review of new outcomes by various cohorts which paraspeckles are established around a long nuclear noncoding RNA (ncRNA), NEAT 1. The result has enhanced the knowledge of functional capabilities of the long ncRNA as well as opening up the feasibility of additional nuclear bodies being established in the same manner (Wilusz, 2016).   

These are a comparatively newly acknowledged sub-nuclear body. They were unraveled when the putative nucleolar protein was detected to localize to nucleoplasmic foci which never overlapped directly with the markers for any particular known sub-nuclear structure (Sadakierska-Chudy and Filip, 2015). Such foci were then named paraspeckles since they stood observed in the interchromatin spaces adjacent to, yet unique from, the speckles of nuclear that stay enriched in splicing factors as seen in the figure below:

The new protein stood named PSPC1, and it has since been the standard marker utilized in the identification of paraspeckles. The paraspeckles are limited to mammalian nuclei as well as observed in the primary and transformed cells lines, tissues, fibroblasts as well as tumorigenic biopsies. They are further dynamic structures; for example, paraspeckles are absent in human embryonic stem cells (Lee, Passon, Hennig, Fox, and Bond,  2011). However, they solely appear under differentiation. Such bodies are 0.5 to 1.0um in size. Their quantities differ both within cell populations as well as depending on the kind of the cell (Whitworth et al, 2017).  For instance, Hela entails thirteen to seventeen paraspeckles a nucleus, while NIH3T3 show between five and ten foci nucleus. At the level of EM, paraspeckles markers label unique nuclear structures which are electron dense as well as rich in the RNA. Such transmission EM also called TEM paraspeckles equates to the interchromatin granule-linked zones (IGAZs). 

Cell Nucleus Organization

The IGAZs are electron-dense fibrillary structures found near the mentioned interchromatin nuclear/granule speckles, whose functions had stood unknown since the early 1990s when they were identified. Presently, paraspeckles are believed to entail a small number of proteins with recorded roles in transcription as well as or RNA processing. Nevertheless, paraspeckles don’t overlap straight with the active transcription sites, based on the bromo-UTP incorporation measurement, albeit they could still form in connection to certain active genes. However, paraspeckles are linked intimately to transcription as a result of active RNA polymerase II (PoI II) presence alongside newly-established RNA at their respective extreme ends (Grimes et al, 2017).

Presently, paraspeckles protein is described by their localization within the sub-nuclear foci with the mammalian DBHS member. There are three members including protein family that entails PSPC1; P54NRB or NONO, or PSF/SFOQ. Such DBHS protein family members remain the most probed intrinsic protein constituent of the paraspeckles (Ahmed, Vélaz, Rosemann,  Gilbertz, and Scherthan, 2017). The documented interactions between the three members of such a family indicate they are in existence either as homo- or heterodimers within the vivo. Such protein share more than fifty percent sequence identity in 2 N-terminal RNP-kind RNA acknowledgment motifs as well as C-terminal coiled-coil domain. Among such RNP-kind RNA acknowledgement motifs alongside the coiled-coil domain that mediates dimerization are essential for the PSPC1 to remain targeted to paraspeckles (Fortini, 2014). The DBHS proteins remain dynamic within nucleus; they are cycling between nucleoplasm, paraspeckles as well as nucleolus within normal conditions as well as accumulating under structures of perinucleolar cap when the RNA PoI II transcription remains inhibited (Duvignaud et al, 2016).

Such a latter outcome explicates the PSPC1 discovery in nucleolar proteome (Knott et al, 2015). The two extremely proteins of DHBS knockdown especially the P54NRB/NONO alongside PSF/SFPQ within the cells of HeLa leads to the paraspeckles loss. In comparison, the less abundant protein of DHBS called PSPC1 knockdown shows no effect on these paraspeckles. Therefore, incredibly expressed DBHS protein dimers remain at the center of structural integrity of the paraspeckles. The DBHS protein family has since been implicated in a broad range of functions. They are already demonstrated to bind to single as well as stranded RNA and DNA and co-purified in some distinct complexes, culminating in the catch-all label of the “malfunction nuclear proteins.”     

It has been revealed that SFPQ interacts with some IncRNAs. Apart from NEAT1, many other IncRNAs have been shown to entail SFPQ binding sites in PAR-CLIP. Individually, new evidence has been presented that SFPQ remains binding MALAT1, an IncRNA remarkably expressed as well as standard in cancer. Albeit MALAT1 has failed to localize to paraspeckles, it remains feasible that it works as part of complex with SFPQ outside of the paraspeckles. Provided this new evidence for the overlapping roles for NEAT1_v1 alongside MALAT1 in targeting transcriptionally active genes as well as MALAT1 competing for SAFPQ binding, additional studies probing the mechanism of binding DBHS proteins to MALAT1 would be essential (Bond and Fox, 2009). It has also been shown that SFPQ bind various illnesses linked with RNAS. For example a number of RNAs bound by SFPQ are engaged in infectious illness and cancer pathways. Such a revelation implicates the mentioned proteins in the response of the cell to these illnesses hence interaction between the proteins and the bound RNAs denote a new therapeutic targets. Nevertheless, to therapeutically modulate such interactions in a meaningful manner, additional information is required relating to the role of proteins in binding such RNAs (Mateo et al, 2015).

It has also been shown that SFPQ bind in long first introns, potentially for the stabilization or directing processing of such pre-mRNA (Dash et al, 2016). SFPQ seems to display preference for binding in long first introns, a feature that is further illustrated by two additional paraspeckles as well as neurodegenerative disorder proteins called TDP-43 alongside FUS, which bind as well as stabilize such transcripts (Wani et al, 2015). The SFPQ binding’s effect in long first introns has never been established; they could stabilize such transcripts co-transcriptionally. Future studies proving the role of SFPQ in the regulation of mRNAs with long first introns would remain interesting, nevertheless, it could be more helpful to carry out such experiments in the neuronal cell line because of the association with neurobiology for additional proteins which bind long first introns (Passon, 2012).  

The DBHS proteins remain substrates for many post-translation modification as observed in figure 2 above. The SFPQ phosphorylation by Protein Kinase C bars it from attaching to the RNA. However, it stimulate is correlation with the ss alongside dsDNA promoting formation of D-loop. In comparison, Mnk1 and Mnk2 selectively phosphorylate SFPQ at Ser 283 and Ser8, proximal to RRM1, improving RNA binding to 3 UTR of TNF –alpha. In the T-cells GSK3 phosphorylates SFPQ at T687 promoting the interaction with TRAP150, inhibiting SFPQ from binding to the CD45 pre-mRNA. The NONO is further phosphorylated in region proximal to coiled-coil domain (T453, T430 and T412) in the course of mitosis. The T-P motifs phosphorylation gives binding sites for peptidyprolyl isomerase or Pin1 which could result in subsequent conformational alterations of this region (Smogorzewska et al, 2016).

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