Alternative titles; symbols
HGNC Approved Gene Symbol: POLR2A
Cytogenetic location: 17p13.1 Genomic coordinates (GRCh38): 17:7,484,366-7,514,616 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 17p13.1 | Neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities | 618603 | Autosomal dominant | 3 |
DNA-dependent RNA polymerase II (EC 2.7.7.6), a complex multisubunit enzyme, is responsible for the transcription of protein-coding genes. It is composed of 10 to 14 subunits ranging in size from 220 to 10 kD. POLR2A encodes the 220-kD subunit. RNA polymerase II interacts with the promoter regions of genes as well as with a variety of elements and transcription factors to determine essentially all of the parameters that govern transcription, e.g., tissue and development specificity, stress response, etc.
Dynamic phosphorylation and dephosphorylation of 3 serines within a heptapeptide sequence repeated 52 times in the POLR2A C-terminal domain (CTD) and other CTD modifications specify recruitment of proteins that regulate various transcription-related events (Ni et al., 2011).
Cho et al. (1985) isolated genomic sequences for the large subunit of human RNA polymerase II (pol II). Sequences homologous to Drosophila pol II large subunit sequences were present in single copy.
Wintzerith et al. (1992) cloned and sequenced the complete human gene for POLR2A and symbolized it RPBh1. The deduced 1,970-amino acid protein contains 10 conserved domains, designated A through J, a putative C2H2-type zinc-binding element, and a C-terminal domain (CTD) containing 52 copies of a heptapeptide repeat.
Mita et al. (1995) also cloned and sequenced the human POLR2A gene and referred to it as RpIILS. The deduced amino acid sequence was identical to that reported by Wintzerith et al. (1992). The sequence of the 5-prime flanking region is approximately 84% identical to the mouse gene (vs 90% for the coding regions).
Crystal Structure
Cramer et al. (2000) derived a backbone model of a 10-subunit yeast RNA polymerase II using x-ray diffraction data extending to 3-angstrom resolution. All 10 subunits exhibited a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the 3 eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5 (180664), and Rpb9 (180662), that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2 (180661), and Rpb6 (604414), may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.
Cramer et al. (2001) determined the structure of a 10-subunit yeast RNA polymerase II (lacking 2 small subunits dispensable for transcription) derived from 2 crystal forms at 2.8- and 3.1-angstrom resolution. Comparison of the structures reveals a division of the polymerase into 4 mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8-angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Gnatt et al. (2001) determined the crystal structure of RNA polymerase II in the act of transcription at 3.3-angstrom resolution. They observed duplex DNA entering the main cleft of the enzyme and unwinding before the active site. Nine basepairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3-prime end of the RNA in the nucleotide addition site. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, 'abortive cycling' during transcription initiation, and RNA and DNA translocation during transcription elongation.
Bushnell et al. (2004) reported the crystal structure of RNA polymerase II with general transcription factor IIB (TFIIB; 189963) at 4.5-angstrom resolution. The structure reveals 3 features crucial for transcription initiation: an N-terminal zinc ribbon domain of TFIIB that contacts the dock domain of the polymerase, near the path of RNA exit from a transcribing enzyme; a 'finger' domain of TFIIB that is inserted into the polymerase active center; and a C-terminal domain, whose interaction with both the polymerase and with a TATA box-binding protein-promoter DNA complex orients the DNA for unwinding and transcription. TFIIB stabilizes an early initiation complex, containing an incomplete RNA-DNA hybrid region. It may interact with the template strand, which sets the location of the transcription start site, and may interfere with RNA exit, which leads to abortive initiation or promoter escape. Westover et al. (2004) determined the structure of an RNA polymerase II-transcribing complex in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of protein loops in a strand/loop network. Westover et al. (2004) concluded that formation of the network must occur in the transition from abortive initiation to promoter escape.
Meinhart and Cramer (2004) described the structure of a ser2-phosphorylated C-terminal domain peptide of RNA polymerase II bound to the C-terminal domain-interacting domain of PCF11 (608876). The C-terminal domain motif of ser2-pro3-thr4-ser5 forms a beta turn that binds to a conserved groove in the C-terminal domain-interacting domain of PCF11. The ser2 phosphate group does not make direct contact with PCF11, but may be recognized indirectly because it stabilizes the beta turn with an additional hydrogen bond. Iteration of the peptide structure results in a compact beta spiral model of the C-terminal domain. Meinhart and Cramer (2004) suggested that during the mRNA transcription processing cycle, compact spiral regions in the C-terminal domain are unraveled and regenerated in a phosphorylation-dependent manner.
Wang et al. (2009) reported the crystal structure of RNA polymerase II in the third state, the reverse translocated or 'backtracked' state. The defining feature of the backtracked structure is a binding site for the first backtracked nucleotide. This binding site is occupied in case of nucleotide misincorporation in the RNA or damage to the DNA, and is termed the 'P' site because it supports proofreading. The predominant mechanism of proofreading is the excision of a dinucleotide in the presence of the elongation factor SII (TFIIS; see 604784). Structure determination of a cocrystal with TFIIS revealed a rearrangement whereby cleavage of the RNA may take place.
Kostrewa et al. (2009) presented the crystal structure of the complete Pol II-B complex at 4.3-angstrom resolution, and complementary functional data. The results indicated the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active center cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the B-linker that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the B-reader that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and the B-linker, respectively, to trigger B release and elongation complex formation.
Liu et al. (2010) developed a crystal structure of the RNA polymerase II-TFIIB complex at 3.8-angstrom resolution obtained under different solution conditions from the structure obtained by Bushnell et al. (2004) and complementary with it. The crystal structure revealed the carboxy-terminal region of TFIIB, located above the polymerase active center cleft, but showing none of the B finger. In the new structure, the linker between the amino- and carboxyl-terminal regions can also be seen, snaking down from above the cleft toward the active center.
Quantitative Mass Spectrometry
One of the primary goals of proteomics is the description of the composition, dynamics, and connections of the multiprotein modules that catalyze a wide range of biologic functions in cells (Hartwell et al., 1999). The yeast 2-hybrid (Y2H) method is designed to detect binary interactions between proteins in the nucleus of a yeast cell. A limitation of this technique is that it does not detect protein-protein interactions in the context of their physiologic environment. Another approach for the analysis of protein complexes involves the use of affinity chromatography for isolation or enrichment of complexes, followed by mass spectrometric identification of the constituent proteins. Ranish et al. (2003) described a generic strategy for determining the specific composition, changes in composition, and changes in abundance of protein complexes. It was based on the use of isotope-coded affinity tag (ICAT) reagents and mass spectrometry to compare the relative abundances of tryptic peptides derived from suitable pairs of purified or partially purified protein complexes. In a first application, the genuine protein components of a large RNA polymerase II (Pol II) preinitiation complex (PIC) were distinguished from a background of copurifying proteins by comparing the relative abundances of peptides derived from a control sample and the specific complex that was purified from nuclear extracts by a single-step promoter DNA affinity procedure. This was the first time that a fully assembled RNA Pol II PIC had been comprehensively analyzed. The quantitative mass spectrometry technique provided for the first time a detailed description of the partially purified core Pol II complex and led to the detection of potential new components of this extensively studied complex.
Lehmann et al. (2007) showed the intrinsic RNA-dependent RNA polymerase (RdRP) activity of Pol II with only pure polymerase, an RNA template-product scaffold, and nucleoside triphosphates (NTPs). Crystallography revealed the template-product duplex in the site occupied by the DNA-RNA hybrid during transcription. RdRP activity resided at the active site used during transcription, but it was slower and less processive than DNA-dependent activity. RdRP activity was also obtained with part of the hepatitis delta virus (HDV) antigenome. The complex of transcription factor IIS (604784) with Pol II could cleave one HDV strand, create a reactive stem loop in the hybrid site, and extend the new RNA 3-prime end. Short RNA stem loops with a 5-prime extension sufficed for activity, but their growth to a critical length appeared to impair processivity. Lehmann et al. (2007) concluded that the RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes.
Kornberg and Lorch (1991) discussed the mechanism by which genes become accessible for transcription, particularly the final stages of the process in which activator proteins and the transcription machinery confront the nucleosome and also specifically in relation to transcription by RNA pol II.
Buratowski (1994) reviewed 'the basics of basal transcription by RNA polymerase II.' For this enzyme to transcribe a gene, the authors commented that an array of over 20 proteins must be assembled at its promoter. Buratowski (1994) reviewed progress in identifying and purifying these transcription factors, as well as cloning the genes that encode them.
Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA pol II, but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on PolB II-transcribed genes is incompletely understood. Bregman et al. (1996) demonstrated that a fraction of the large subunit of Pol II (PolIILS) is ubiquitinated after exposing cells to UV radiation or cisplatin, but not to several other DNA-damaging agents. This novel covalent modification of PolIILS occurs within 15 minutes of exposing cells to UV-radiation and persists for about 8 to 12 hours. Ubiquitinated PolIILS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of PolIILS is deficient in fibroblasts from persons with either Cockayne syndrome type A (CS-A; 216400) or type B (CS-B; see 133540). In both of these disorders transcription-coupled repair is disrupted. UV-induced ubiquitination of PolIILS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggested that ubiquitination of PolIILS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription, a possibility that had been suggested on other grounds.
The monoclonal antibody CC-3 recognizes a phosphodependent epitope on a 255-kD nuclear matrix protein (p255) shown to associate with spliceosome complexes. Vincent et al. (1996) showed that p255 represents a highly phosphorylated form of Pol II large subunit (IIo) that physically associates with spliceosomes. They suggested that IIo is involved in coupling transcription with RNA processing.
High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation. Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodeling factors. Reinitiation is thought to occur through a different pathway. After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex. Yudkovsky et al. (2000) described the isolation of a reinitiation intermediate in yeast that includes transcription factors TFIID (see 313650), TFIIA (see 600520), TFIIH (see 189972), TFIIE (see 189962), and Mediator (see 602984). This intermediate can act as a scaffold for formation of a functional reinitiation complex. Formation of this scaffold is dependent on ATP and TFIIH. In yeast, the scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.
In a yeast 2-hybrid screen to identify proteins that interact with the phosphorylated C-terminal domain (CTD) of POLR2A, Bourquin et al. (1997) identified PPIG (606093). Using GST fusion proteins, they demonstrated direct interaction between PPIG and the CTD of POLR2A.
Dye and Proudfoot (2001) performed in vivo analysis of transcriptional termination for the human beta-globin gene (141900) and demonstrated cotranscriptional cleavage (CoTC). This primary cleavage event within beta-globin pre-mRNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA pol II. Teixeira et al. (2004) showed that the CoTC process in the human beta-globin gene involves an RNA self-cleaving activity. They characterized the autocatalytic core of the CoTC ribozyme and showed its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3-prime flanking regions of other primate beta-globin genes. Functionally, it resembles the 3-prime processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. Teixeira et al. (2004) predicted that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide an entry point for exonucleases involved in mRNA maturation, turnover, and, in particular, transcriptional termination.
Using a variety of RNA-binding assays, Kaneko and Manley (2005) showed that the CTD of mammalian POLR2A interacted with RNA in a sequence-specific manner in vitro and in vivo. The CTD-binding consensus sequence downstream of a polyadenylation signal suppressed mRNA 3-prime end formation and transcription termination. In vitro assays indicated that the inhibition of processing is CTD dependent.
In yeast, the Paf1 complex interacts with DNA polymerase II and is involved in multiple aspects of histone methylation. By immunoprecipitation, Rozenblatt-Rosen et al. (2005) determined the components of the human PAF1 complex (see 610506). The immunoprecipitate also contained POLR2A that was unphosphorylated, phosphorylated on ser5, or phosphorylated on ser2, suggesting that the PAF1 complex may be involved in both initiation and elongation.
Cells use transcription-coupled repair to efficiently eliminate DNA lesions such as ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs). Brueckner et al. (2007) presented the structure-based mechanism for the first step in eukaryotic transcription-coupled repair, CPD-induced stalling of RNA polymerase (Pol) II. A CPD in the transcribed strand slowly passes a translocation barrier and enters the polymerase active site. The CPD 5-prime thymine then directs uridine misincorporation into mRNA, which blocks translocation. Artificial replacement of the uridine by adenosine enables CPD bypass; thus, Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. Brueckner et al. (2007) concluded that this is consistent with nonallosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II.
The carboxy-terminal domain (CTD) of the large subunit of mammalian polymerase II consists of 52 repeats of a consensus heptapeptide tyr-ser-pro-thr-ser-pro-ser. Differential phosphorylation of ser2 and ser5 at the 5-prime and 3-prime regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex (Chapman et al., 2007). Egloff et al. (2007) showed that mutation of ser7 to ala causes a specific defect in snRNA gene expression. They also presented evidence that phosphorylation of ser7 facilitates interaction with the snRNA gene-specific integrator complex. Egloff et al. (2007) concluded that their findings assigned a biologic function to this amino acid and highlighted a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.
Using monoclonal antibodies, Chapman et al. (2007) revealed ser7 phosphorylation of RNA polymerase-2 on transcribed genes. This position did not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where ser7 was substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTDs. Chapman et al. (2007) concluded that restriction of ser7 epitopes to the linker-proximal regions limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
To study nutritional control of C. elegans in larval development, Baugh et al. (2009) analyzed growth and gene expression profiles during L1 arrest and recovery. Larvae that were fed responded relatively slowly to starvation compared with the rapid response of arrested larvae to feeding. Chromatin immunoprecipitation of RNA polymerase II followed by deep sequencing showed that during L1 arrest, Pol II continued transcribing starvation-response genes, but the enzyme accumulated on the promoters of growth and development genes. In response to feeding, promoter accumulation decreased, and elongation and mRNA levels increased. Therefore, Baugh et al. (2009) concluded that accumulation of Pol II at promoters anticipates nutritionally-controlled gene expression during C. elegans development.
Natalizio et al. (2009) noted that the CTD of POLR2A is not present in homologous subunits of RNA polymerases I and III. They found that fusing the CTD of POLR2A to POLR3A (614258) did not enhance the cotranscriptional pre-mRNA splicing or capping activity of POLR3A in transfected cells. Furthermore, fusing the CTD of POLR2A to the bacteriophage T7 RNA did not enhance pre-mRNA splicing or capping in vitro or in vivo. Natalizio et al. (2009) proposed that efficient coupling of transcription to pre-mRNA processing requires not only the phosphorylated CTD of POLR2A but also other RNA polymerase II-specific subunits or associated factors.
Kasowski et al. (2010) examined genomewide differences in transcription factor binding in several humans and a single chimpanzee by using chromatin immunoprecipitation followed by sequencing. They mapped the binding sites of RNA polymerase II and NF-kappa-B (see 164011) in 10 lymphoblastoid cell lines and found that 25% and 7.5% of the respective binding regions differed between individuals. Binding differences were frequently associated with SNPs and genomic structural variants, and these differences were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, the results of comparing PolII binding between humans and chimpanzee suggested extensive divergence in transcription factor binding.
The carboxy-terminal domain of RNA polymerase II in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Sims et al. (2011) showed that the carboxy-terminal domain of RNA polymerase II is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase-1 (CARM1; 603934). Although methylation at R1810 is present on the hyperphosphorylated form of RNA polymerase II in vivo, ser2 or ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1 -/- mouse embryo fibroblasts. Sims et al. (2011) concluded that carboxy-terminal domain methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNA polymerase II-associated machinery recruited to distinct gene types.
The RNA polymerase II largest subunit contains a CTD with up to 52 Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) consensus repeats. Serines 2, 5, and 7 are known to be phosphorylated, and these modifications help to orchestrate the interplay between transcription and processing of mRNA precursors. Hsin et al. (2011) provided evidence that phosphorylation of CTD Thr(4) residues is required specifically for histone mRNA 3-prime end processing, functioning to facilitate recruitment of 3-prime processing factors to histone genes. Like Ser(2), Thr(4) phosphorylation requires the CTD kinase CDK9 (603251) and is evolutionarily conserved from yeast to human. Hsin et al. (2011) concluded that their data illustrate how a CTD modification can play a highly specific role in facilitating efficient gene expression.
Using affinity chromatography and tandem mass spectrometry with HEK293 cells to isolate RNA polymerase II-interacting proteins, Ni et al. (2011) identified RPRD1A (610347), RPRD1B (614694), and RPRD2 (614695), in addition to RECQL5 (603781), GRINL1A (606485), and the putative RNA polymerase II phosphatase RPAP2 (611476). RPRD1A and RPRD1B accompanied RNA polymerase II from promoter regions to 3-prime UTRs during transcription in vivo. RPRD1A and RPRD1B coprecipitated with the C-terminal domain of POLR2A when it was serine phosphorylated, but not when it was unphosphorylated. Overexpression of RPRD1A or RPRD1B reduced the amount of serine-phosphorylated POLR2A associated with a target gene.
Zhao et al. (2016) showed that a carboxy-terminal domain (CTD) arginine (R1810 in human) that is conserved across vertebrates is symmetrically dimethylated (me2s). This R1810me2s modification requires PRMT5 (604045) and recruits the Tudor domain of SMN (600354). SMN interacts with senataxin (SETX; 608465). Because POLR2A R1810me2s and SMN, like senataxin, are required for resolving RNA-DNA hybrids created by RNA polymerase II that form R-loops in transcription termination regions, Zhao et al. (2016) proposed that R1810me2s, SMN, and senataxin are components of an R-loop resolution pathway.
Abraham et al. (2020) showed that RNA Pol II inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. This shield prevents Pol I (see 616404) from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma, or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1 (604123), eGFP, and dCas9, which the authors referred to as 'RED-LasRR.' Abraham et al. (2020) revealed a nucleolar Pol II-dependent mechanism that drives ribosome biogenesis, identified disease-associated disruption of nucleoli by noncoding RNAs, and established locus-targeted R-loop modulation. Abraham et al. (2020) concluded that their findings revised theories of labor division between the major RNA polymerases, and identified nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.
Mita et al. (1995) determined that the POLR2A gene contains 29 exons and spans about 32 kb of DNA. Intron size varies considerably between species, largely as a consequence of Alu and B1 insertions in the human and mouse genes, respectively. The 5-prime flanking region of POLR2A contains several binding sites for the transcription factor Sp1 (189906), a CCAAT sequence, and a sequence homologous to a heat-shock element.
Cannizzaro et al. (1986) assigned the human gene to the distal portion of the short arm of chromosome 17 (17pter-p12) by in situ hybridization and Southern analysis of DNA from somatic cell hybrids.
Pravtcheva et al. (1986) mapped the gene encoding the largest subunit of RNA pol II to mouse chromosome 11 by Southern blot analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This is another example of the homology of mouse chromosome 11 and human chromosome 17. Somatic cell hybrid studies by vanTuinen and Ledbetter (1987) narrowed the assignment to 17p13.105-p12. Using in situ hybridization, Acker et al. (1994) likewise mapped the POLR2A gene to 17p13.
Liu et al. (2015) demonstrated that genomic deletion of TP53 (191170) frequently encompasses essential neighboring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. The authors identified POLR2A as such a gene that is almost always codeleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by alpha-amanitin. Liu et al. (2015) analyzed the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases, which revealed that expression levels of POLR2A are tightly correlated with its gene copy numbers in human colorectal cancer (CRC; 114500). Suppression of POLR2A with alpha-amanitin or siRNAs selectively inhibits the proliferation, survival, and tumorigenic potential of CRC cells with hemizygous TP53 loss in a p53-independent manner. Clinical applications of alpha-amanitin had been limited owing to its liver toxicity; however, Liu et al. (2015) found that alpha-amanitin-based antibody-drug conjugates (Moldenhauer et al., 2012) are highly effective therapeutic agents with reduced toxicity. Liu et al. (2015) showed that low doses of alpha-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM; 185535) antibody led to complete tumor regression in mouse models of human CRC with hemizygous deletion of POLR2A.
In 11 unrelated individuals with neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified de novo heterozygous mutations in the POLR2A gene (see, e.g., 180660.0001-180660.0005). The patients were ascertained through the GeneMatcher initiative, and the mutations were identified through whole-exome or whole-genome studies. Using a combination of assessments and techniques, the authors classified these 11 mutations as 'probably disease-causing.' In vitro studies of some of the corresponding mutations in yeast and HeLa cells showed variable results, with most causing poor growth and/or reduced cell viability, suggesting a pathogenic effect. Most of the missense mutations centered around the catalytic site, but did not interfere with formation of the Pol II complex; these findings suggested a dominant-negative effect of the missense variants. In contrast, the nonsense, frameshift, or in-frame deletion mutations were predicted to have a loss-of-function effect resulting in haploinsufficiency. In general, patients with missense variants had a more severe phenotype compared to those with loss-of-function mutations. Haijes et al. (2019) suggested that the presence of a malfunctioning species of pol II resulting from missense mutations is more detrimental than reduced availability of pol II resulting from loss-of-function mutations. The authors speculated that aberrant pol II enzymes may be able to assemble properly at transcription start sites, but may impair subsequent elongation of nascent RNA causing increased error rates and abnormal release dynamics. Haijes et al. (2019) also identified 5 additional patients with an overlapping phenotype associated with de novo heterozygous POLR2A variants; however, these variants were classified as 'possibly pathogenic' (4 cases) or unknown (1 case).
From a strain of fetal human lung diploid fibroblasts, Buchwald and Ingles (1976) isolated clones resistant to the cytotoxic action of alpha-amanitin. The resistant clones were recovered at a frequency of 5 x 10(-8) following mutagenesis with ethyl methanane sulfonate. The clones retained the resistant phenotype after propagation in drug-free medium. The amanitin sensitivity of RNA pol II purified from the mutant cells suggested the presence of 2 forms of the enzyme, one similar to that in wildtype cells and the second with increased resistance to alpha-amanitin inhibition. Thus, alpha-amanitin resistance behaves as a dominant. It is dominant in Chinese hamster and rat cells, also. Amanitin is a bicyclic octapeptide produced by the mushroom Amanita phaloides. In Drosophila, alpha-amanitin resistance mutations are located in the genes for the large subunit of RNA pol II. The same is presumably true for amanitin-resistance in human cells. Transfection experiments referred to by Cannizzaro et al. (1986) supported this idea. A relationship to homeotic genes (142960) and other genes on 17p was raised, as well as a possible etiologic role of mutation in RPOL2 in the Miller-Dieker syndrome (247200). Drosophila mutants of RNA pol II show developmental abnormalities.
In a 4-year-old boy (patient 2) with a severe form of neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified a de novo heterozygous c.1370T-C transition (c.1370T-C, NM_000937.4) in the POLR2A gene, resulting in an ile457-to-thr (I457T) substitution at a conserved residue in the catalytic site. In vitro studies of the corresponding mutation in yeast resulted in poor growth, suggesting decreased transcriptional fidelity, but expression of the mutation in HeLa cells did not result in decreased cell viability compared to controls. Interactome analysis indicated that the variant could still interact with other subunits to form the pol II complex. The authors suggested a dominant-negative effect.
In a 17-year-old boy (patient 5) with a mild form of neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified a de novo heterozygous c.2098C-T transition (c.2098C-T, NM_000937.4) in the POLR2A gene, resulting in a gln700-to-ter (Q700X) substitution. In vitro studies using a similar mutation (L812X) in HeLa cells resulted in decreased cell viability, and interactome analysis indicated that the variant could not interact with most other pol II subunits. The authors postulated a loss-of-function effect and haploinsufficiency.
In a 13-year-old girl (patient 6) with a mild form of neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified a de novo heterozygous c.2203C-T transition (c.2203C-T, NM_000937.4) in the POLR2A gene, resulting in a gln735-to-ter (Q735X) substitution. In vitro studies using a similar mutation (L812X) in HeLa cells resulted in decreased cell viability, and interactome analysis indicated that the variant could not interact with most other pol II subunits. The authors postulated a loss-of-function effect and haploinsufficiency.
In a 4-year-old girl (patient 7) with profound form of neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified a de novo heterozygous c.2207C-T transition (c.2207C-T, NM_000937.4) in the POLR2A gene, resulting in a thr736-to-met (T736M) substitution at a conserved residue in the quay domain. In vitro studies of the corresponding mutation in yeast resulted in poor growth, suggesting decreased transcriptional fidelity, and expression of the mutation in HeLa cells resulted in decreased cell viability compared to controls. Interactome analysis indicated that the variant could still interact with other subunits to form the pol II complex. The authors suggested a dominant-negative effect.
In a 4-year-old girl (patient 12) with a mild form of neurodevelopmental disorder with hypotonia and variable intellectual and behavioral abnormalities (NEDHIB; 618603), Haijes et al. (2019) identified a de novo heterozygous c.3371T-C transition (c.3371T-C, NM_000937.4) in the POLR2A gene, resulting in a leu1124-to-pro (L1124P) substitution at a conserved residue in the quay domain. In vitro studies of the corresponding mutation in yeast resulted in poor growth, suggesting decreased transcriptional fidelity, and expression of the mutation in HeLa cells resulted in decreased cell viability compared to controls. Interactome analysis indicated that the variant could still interact with other subunits to form the pol II complex. The authors suggested a dominant-negative effect.
Abraham, K. J., Khosraviani, N., Chan, J. N. Y., Gorthi, A., Samman, A., Zhao, D. Y., Wang, M., Bokros, M., Vidya, E., Ostrowski, L. A., Oshidari, R., Pietrobon, V., and 13 others. Nucleolar RNA polymerase II drives ribosome biogenesis. Nature 585: 298-302, 2020. [PubMed: 32669707] [Full Text: https://doi.org/10.1038/s41586-020-2497-0]
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