Decapping Activity Research Articles

Nudt3 is an mRNA decapping enzyme that modulates cell migration

Removal of the 5′-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.

The human decapping scavenger enzyme DcpS modulates microRNA turnover.

The decapping scavenger enzyme DcpS is known for its role in hydrolyzing the cap structure following mRNA degradation. Recently, we discovered a new function in miRNA degradation activation for the ortholog of DcpS in C. elegans. Here we show that human DcpS conserves its role in miRNA turnover. In human cells, DcpS is a nucleocytoplasmic shuttling protein that activates miRNA degradation independently of its scavenger decapping activity in the cytoplasmic compartment. We also demonstrate that this new function for DcpS requires the contribution of the 5′-3′ exonuclease Xrn2. Our findings support a conserved role of DcpS as a modulator of miRNA turnover in animals.

Geminivirus Activates ASYMMETRIC LEAVES 2 to Accelerate Cytoplasmic DCP2-Mediated mRNA Turnover and Weakens RNA Silencing in Arabidopsis.

Aberrant viral RNAs produced in infected plant cells serve as templates for the synthesis of dsRNAs. The derived virus-related small interfering RNAs (siRNA) mediate cleavage of viral RNAs by post-transcriptional gene silencing (PTGS), thus blocking virus multiplication. Here, we identified ASYMMETRIC LEAVES2 (AS2) as a new component of plant P body complex which mediates mRNA decapping and degradation. We found that AS2 promotes DCP2 decapping activity, accelerates mRNA turnover rate, inhibits siRNA accumulation and functions as an endogenous suppressor of PTGS. Consistent with these findings, as2 mutant plants are resistant to virus infection whereas AS2 over-expression plants are hypersensitive. The geminivirus nuclear shuttle protein BV1 protein, which shuttles between nuclei and cytoplasm, induces AS2 expression, causes nuclear exit of AS2 to activate DCP2 decapping activity and renders infected plants more sensitive to viruses. These principles of gene induction and shuttling of induced proteins to promote mRNA decapping in the cytosol may be used by viral pathogens to weaken antiviral defenses in host plants.

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain.

Decapping commits an mRNA to complete degradation and promotes general 5′ to 3′ decay, nonsense-mediated decay (NMD), and transcript-specific degradation. In Saccharomyces cerevisiae, a single decapping enzyme composed of a regulatory subunit (Dcp1) and a catalytic subunit (Dcp2) targets thousands of distinct substrate mRNAs. However, the mechanisms controlling this enzyme's in vivo activity and substrate specificity remain elusive. Here, using a genetic approach, we show that the large C-terminal domain of Dcp2 includes a set of conserved negative and positive regulatory elements. A single negative element inhibits enzymatic activity and controls the downstream functions of several positive elements. The positive elements recruit the specific decapping activators Edc3, Pat1, and Upf1 to form distinct decapping complexes and control the enzyme's substrate specificity and final activation. Our results reveal unforeseen regulatory mechanisms that control decapping enzyme activity and function in vivo, and define roles for several decapping activators in the regulation of mRNA decapping.

Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping.

Recombinant modified vaccinia virus Ankara (MVA) has been used to deliver vaccine candidate antigens against infectious diseases and cancer. MVA is a potent viral vector for inducing high magnitudes of antigen-specific CD8+ T cells; however the cellular immune responses to a recombinant antigen in MVA could be further enhanced by increasing transgene expression. Previous reports showed the importance of utilizing an early poxviral promoter for increasing transgene expression and therefore enhancing cellular immune responses. However, the vaccinia D10 decapping enzyme is reported to target and decap vaccinia virus early transcripts – a mechanism that could limit the usefulness of early promoters in MVA viral vectors if this enzyme shows the same activity in this closely related virus. Therefore, we attempted to increase transgene expression in recombinant MVA by inserting the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) upstream of a transgene sequence that is controlled by the B8R early promoter, and assessed D10 enzyme decapping activity in MVA. The aim of the IRES element was to initiate translation of the transgene transcript (after the removal of the cap structure by the D10 decapping protein) in a cap-independent manner. Here, we report that overexpression of the D10 decapping protein, in trans, in MVA reduced growth and transgene expression; however, the IRES element was not able to compensate for the negative effect of the D10 decapping protein. Recombinant MVA with EMCV IRES induced levels of both gene expression and transcription that were similar to the control recombinant MVA, encoding the same transgene but without the IRES element. Both viruses were tested in BALB/c mice and induced similar magnitudes of epitope-specific CD8+ T cells. This work indicates that the MVA version of the D10 decapping enzyme, overexpressed using a plasmid, is functional, but its negative effect on transgene expression by recombinant MVA cannot be overcome by the use of the EMCV IRES inserted upstream of the transgene initiation codon.

DNA3'pp5'G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition.

DNA3′pp5′G caps synthesized by the 3′-PO4/5′-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3′-OH ends. Aprataxin, an enzyme that repairs A5′pp5′DNA ends formed during abortive ligation by classic 3′-OH/5′-PO4 ligases, is also a DNA 3′ de-capping enzyme, converting DNAppG to DNA3′p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3′ de-guanylylation and 5′ de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.

DcpS is a transcript-specific modulator of RNA in mammalian cells.

The scavenger decapping enzyme DcpS is a multifunctional protein initially identified by its property to hydrolyze the resulting cap structure following 3′ end mRNA decay. In Saccharomyces cerevisiae, the DcpS homolog Dcs1 is an obligate cofactor for the 5′-3′ exoribonuclease Xrn1 while the Caenorhabditis elegans homolog Dcs-1, facilitates Xrn1 mediated microRNA turnover. In both cases, this function is independent of the decapping activity. Whether DcpS and its decapping activity can affect mRNA steady state or stability in mammalian cells remains unknown. We sought to determine DcpS target genes in mammalian cells using a cell-permeable DcpS inhibitor compound, RG3039 initially developed for therapeutic treatment of spinal muscular atrophy. Global mRNA levels were examined following DcpS decapping inhibition with RG3039. The steady-state levels of 222 RNAs were altered upon RG3039 treatment. Of a subset selected for validation, two transcripts that appear to be long noncoding RNAs HS370762 and BC011766, were dependent on DcpS and its scavenger decapping catalytic activity and referred to as DcpS-responsive noncoding transcripts (DRNT) 1 and 2, respectively. Interestingly, only the increase in DRNT1 transcript was accompanied with an increase of its RNA stability and this increase was dependent on both DcpS and Xrn1. Importantly, unlike in yeast where the DcpS homolog is an obligate cofactor for Xrn1, stability of additional Xrn1 dependent RNAs were not altered by a reduction in DcpS levels. Collectively, our data demonstrate that DcpS in conjunction with Xrn1 has the potential to regulate RNA stability in a transcript-selective manner in mammalian cells.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

Role of cytoplasmic deadenylation and mRNA decay factors in yeast apoptosis.

Strains of Saccharomyces cerevisiae lacking factors involved in 5' to 3' mRNA decay pathway (DCP1, DCP2, DHH1, PAT1, LSM1 and LSM4) exhibit caspase-dependent apoptosis and accelerated chronological aging. In the present study, yeast strains lacking mRNA decapping activation factors (DCP2 and LSM1), cytoplasmic exosome function (SKI2) or cytoplasmic deadenylases (double deletion of CCR4 and PAN2) showed typical markers of eukaryotic apoptosis such as increased cellular reactive oxygen species levels, externalization of phosphatidyl serine, chromatin fragmentation, enhanced caspase gene (YCA1) expression and protein activity in mid-log phase cultures. The transcript levels of negative regulators of mRNA decapping (eIF4E and Pab1) were considerably elevated in strains defective in cytoplasmic deadenylation and reduced in strains lacking cytoplasmic 3' to 5' exosome function or decapping activators. Among the yeast strains studied, lsm1Δ and ccr4Δpan2Δ mutants displayed strongest apoptotic phenotype followed by mutants lacking DCP2 or SKI2. Among yeast strains exhibiting deadenylation defects, slight apoptotic phenotype was observed in ccr4Δ mutants and cell death markers imperceptible in pan2Δ mutants.

Role of the DHH1 gene in the regulation of monocarboxylic acids transporters expression in Saccharomyces cerevisiae.

Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170∶301–306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

Structural and functional studies of the decapping exoribonucleases

Recent studies showed that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Rai1 possesses a novel decapping activity that can remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Dxo1 also has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, we found that Dxo1 also possesses distributive, 5'-3' exoribonuclease activity, and we named Dxo1 (originally Ydr370C) for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process. Here, we introduce that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5 Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.

Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4-NOT deadenylase complex followed by the removal of the 5' cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4-NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6-CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.

A DDX6-CNOT1 Complex and W-Binding Pockets in CNOT9 Reveal Direct Links between miRNA Target Recognition and Silencing

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.

Yeast Edc3 targets RPS28B mRNA for decapping by binding to a 3' untranslated region decay-inducing regulatory element.

mRNA decapping commits a transcript to complete turnover in eukaryotic cells. In yeast, general mRNA decapping requires the Dcp1/Dcp2 decapping enzyme and a set of decapping activators, including Pat1, Dhh1, Edc3, and the Lsm1-7 complex. The exact function and mode of action of each of these decapping activators in mRNA decapping largely remain elusive. Here, we analyzed the role of Edc3 in the decay of yeast RPS28B mRNA, a pathway triggered by a negative-feedback autoregulatory mechanism. We show that Edc3-mediated RPS28B mRNA decay requires either of two orthologous proteins, Rps28a and Rps28b, expressed from the RPS28A and RPS28B genes, respectively. Contrary to a generally accepted model, we found that Rps28b does not bind to the 3'-untranslated region (UTR) regulatory element in RPS28B mRNA. Instead, Edc3 is directly involved in binding the element, and Rps28b binds Edc3 and regulates its activity. Decay of RPS28B mRNA requires the Lsm and YjeF-N domains of Edc3, but surprisingly, decay of YRA1 pre-mRNA, the only other known substrate of Edc3, requires only the Lsm domain. Collectively, our experiments reveal a new role for Edc3 in mRNA substrate recognition and suggest that this activity is subject to intricate regulation by additional factors, including the Rps28 ribosomal protein.

An AU-rich instability element in the 3'UTR mediates an increase in mRNA stability in response to expression of a dhh1 ATPase mutant.

The DEAD box RNA helicase DHH1 acts as a general repressor of translation and activator of decapping but can also act specifically on individual mRNAs. In trypanosomes, DHH1 overexpression or expression of a dhh1 ATPase mutant, dhh1 DEAD:DQAD, resulted in increased or decreased stability of a small group of mRNAs, mainly encoding developmentally regulated genes. Here, four of the mRNAs affected by dhh1 DEAD:DQAD expression have been analyzed to identify cis-elements involved in dhh1 DEAD:DQAD action. For three mRNAs, the 3′ UTR mediated the change in mRNA level and, in one case, both the 5′ and the 3′ UTR contributed. No responsive elements were detected in the protein coding sequences. One mRNA stabilized by dhh1 DEAD:DQAD expression was analyzed in more detail: deletion or mutation of an AU-rich element in the 3′ UTR resulted in mRNA stabilization in the absence of dhh1 DEAD:DQAD and completely abolished the response to dhh1 DEAD:DQAD. While AU-rich instability elements have been previously shown to mediate mRNA decrease or translational exit by recruitment of DHH1, this is, to our knowledge, the first report of an AU-rich instability element that is responsible for a DHH1 mediated increase in mRNA stability. We suggest a novel model for the selective action of dhh1 on individual mRNAs that is based on the change in the turnover rate of stabilizing or destabilizing RNA binding proteins.

Lsm2 and Lsm3 bridge the interaction of the Lsm1-7 complex with Pat1 for decapping activation

The evolutionarily conserved Lsm1-7-Pat1 complex is the most critical activator of mRNA decapping in eukaryotic cells and plays many roles in normal decay, AU-rich element-mediated decay, and miRNA silencing, yet how Pat1 interacts with the Lsm1-7 complex is unknown. Here, we show that Lsm2 and Lsm3 bridge the interaction between the C-terminus of Pat1 (Pat1C) and the Lsm1-7 complex. The Lsm2-3-Pat1C complex and the Lsm1-7-Pat1C complex stimulate decapping in vitro to a similar extent and exhibit similar RNA-binding preference. The crystal structure of the Lsm2-3-Pat1C complex shows that Pat1C binds to Lsm2-3 to form an asymmetric complex with three Pat1C molecules surrounding a heptameric ring formed by Lsm2-3. Structure-based mutagenesis revealed the importance of Lsm2-3-Pat1C interactions in decapping activation in vivo. Based on the structure of Lsm2-3-Pat1C, a model of Lsm1-7-Pat1 complex is constructed and how RNA binds to this complex is discussed.

HPat a Decapping Activator Interacting with the miRNA Effector Complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.

MiRISC recruits decapping factors to miRNA targets to enhance their degradation

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5′-to-3′ messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5′ cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

Judge, Jury, and Executioner: DXO Functions as a Decapping Enzyme and Exoribonuclease in Pre-mRNA Quality Control

In this issue of Molecular Cell, Jiao et al. (2013) describe the mammalian enzyme DXO, which has pyrophosphohydrolase, decapping, and 5'-3' exoribonuclease activity and functions as an important checkpoint in cotranscriptional capping of RNA polymerase II (Pol II) pre-mRNA transcripts.