Cap Structure Research Articles

TOP1α fine-tunes TOR-PLT2 to maintain root tip homeostasis in response to sugars.

Plant development is highly dependent on energy levels. TARGET OF RAPAMYCIN (TOR) activates the proximal root meristem to promote root development in response to photosynthesis-derived sugars during photomorphogenesis in Arabidopsis thaliana. However, the mechanisms of how root tip homeostasis is maintained to ensure proper root cap structure and gravitropism are unknown. PLETHORA (PLT) transcription factors are pivotal for the root apical meristem (RAM) identity by forming gradients, but how PLT gradients are established and maintained, and their roles in COL development are not well known. We demonstrate that endogenous sucrose induces TOPOISOMERASE1α (TOP1α) expression during the skotomorphogenesis-to-photomorphogenesis transition. TOP1α fine-tunes TOR expression in the root tip columella. TOR maintains columella stem cell identity correlating with reduced quiescent centre cell division in a WUSCHEL RELATED HOMEOBOX5-independent manner. Meanwhile, TOR promotes PLT2 expression and phosphorylates and stabilizes PLT2 to maintain its gradient consistent with TOR expression pattern. PLT2 controls cell division and amyloplast formation to regulate columella development and gravitropism. This elaborate mechanism helps maintain root tip homeostasis and gravitropism in response to energy changes during root development.

Role of Phosphorus-Containing Molecules on the Formation of Nano-Sized Calcium Phosphate for Bone Therapy.

Calcium phosphate (CaP) is the principal inorganic constituent of bone and teeth in vertebrates and has various applications in biomedical areas. Among various types of CaPs, amorphous calcium phosphate (ACP) is considered to have superior bioactivity and biodegradability. With regard to the instability of ACP, the phosphorus-containing molecules are usually adopted to solve this issue, but the specific roles of the molecules in the formation of nano-sized CaP have not been clearly clarified yet. Herein, alendronate, cyclophosphamide, zoledronate, and foscarnet are selected as the model molecules, and theoretical calculations were performed to elucidate the interaction between calcium ions and different model molecules. Subsequently, CaPs were prepared with the addition of the phosphorus-containing molecules. It is found that cyclophosphamide has limited influence on the generation of CaPs due to their weak interaction. During the co-precipitation process of Ca2+ and PO4 3-, the competitive relation among alendronate, zoledronate, and foscarnet plays critical roles in the produced inorganic-organic complex. Moreover, the biocompatibility of CaPs was also systematically evaluated. The DFT calculation provides a convincing strategy for predicting the structure of CaPs with various additives. This work is promising for designing CaP-based multifunctional drug delivery systems and tissue engineering materials.

Autophagy promotes organelle clearance and organized cell separation of living root cap cells in Arabidopsis thaliana.

ABSTRACTThe root cap is a multilayered tissue covering the tip of a plant root that directs root growth through its unique functions, such as gravity sensing and rhizosphere interaction. To maintain the structure and function of the root cap, its constituent cells are constantly turned over through balanced cell division and cell detachment in the inner and outer cell layers, respectively. Upon displacement toward the outermost layer, columella cells at the central root cap domain functionally transition from gravity-sensing cells to secretory cells, but the mechanisms underlying this drastic cell fate transition are largely unknown. Here, using live-cell tracking microscopy, we show that organelles in the outermost cell layer undergo dramatic rearrangements. This rearrangement depends, at least partially, on spatiotemporally regulated activation of autophagy. Notably, this root cap autophagy does not lead to immediate cell death, but is instead necessary for organized separation of living root cap cells, highlighting a previously undescribed role of developmentally regulated autophagy in plants. This article has an associated ‘The people behind the papers’ interview.

RNase H-based analysis of synthetic mRNA 5' cap incorporation.

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5′ cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5′ cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5′ end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5′ cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5′ cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Noncanonical metabolite RNA caps: Classification, quantification, (de)capping, and function.

The 5' cap of eukaryotic mRNA is a hallmark for cellular functions from mRNA stability to translation. However, the discovery of novel 5'-terminal RNA caps derived from cellular metabolites has challenged this long-standing singularity in both eukaryotes and prokaryotes. Reminiscent of the 7-methylguanosine (m7G) cap structure, these noncanonical caps originate from abundant coenzymes such as NAD, FAD, or CoA and from metabolites like dinucleoside polyphosphates (NpnN). As of now, the significance of noncanonical RNA caps is elusive: they differ for individual transcripts, occur in distinct types of RNA, and change in response to environmental stimuli. A thorough comparison of their prevalence, quantity, and characteristics is indispensable to define the distinct classes of metabolite-capped RNAs. This is achieved by a structured analysis of all present studies covering functional, quantitative, and sequencing data which help to uncover their biological impact. The biosynthetic strategies of noncanonical RNA capping and the elaborate decapping machinery reveal the regulation and turnover of metabolite-capped RNAs. With noncanonical capping being a universal and ancient phenomenon, organisms have developed diverging strategies to adapt metabolite-derived caps to their metabolic needs, but ultimately to establish noncanonical RNA caps as another intriguing layer of RNA regulation. This article is categorized under: RNA Processing > Capping and 5' End Modifications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Turnover and Surveillance > Regulation of RNA Stability.

The mTOR/4E-BP1/eIF4E Signalling Pathway as a Source of Cancer Drug Targets.

The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4EBPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signalling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1, and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds.

A Strategy for Gene Knockdown in Dinoflagellates.

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5′-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

Crystal structure of thermally stable homodimeric cytochrome c'-β from Thermus thermophilus.

Cytochrome c'-β is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel β-sheet fold. Here, the crystal structure of cytochrome c'-β from the thermophilic Thermus thermophilus (TTCP-β) is reported at 1.74 Å resolution. TTCP-β has a typical antiparallel β-sheet fold similar to that of cytochrome c'-β from the moderately thermophilic Methylococcus capsulatus (MCCP-β). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-β and MCCP-β, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-β exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-β. Mutational analysis reveals that the increased homodimeric interface area of TTCP-β contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-β loop regions, possibly contribute to the rigid loop structure compared with MCCP-β, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-β, including TTCP-β, are optimized for function under the high-temperature conditions in which the source organisms live.

Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction.

Site-specific chemical modification of mRNA can improve its translational efficiency and stability. For this purpose, it is desirable to develop a complete chemical synthesis method for chemically modified mRNA. The key is a chemical reaction that introduces a cap structure into the chemically synthesized RNA. In this study, we developed a fast and quantitative chemical capping reaction between 5'-phosphorylated RNA and N7-methylated GDP imidazolide in the presence of 1-methylimidazole in the organic solvent dimethyl sulfoxide. It enabled quantitative preparation of capping RNA within 3 h. We prepared chemically modified 107-nucleotide mRNAs, including N6-methyladenosine, insertion of non-nucleotide linkers, and 2'-O-methylated nucleotides at the 5' end and evaluated their effects on translational activity in cultured HeLa cells. The results showed that mRNAs with non-nucleotide linkers in the untranslated regions were sufficiently tolerant to translation and that mRNAs with the Cap_2 structure had higher translational activity than those with the Cap_0 structure.

Design, Synthesis, Bioactivity Evaluation, Crystal Structures, and In Silico Studies of New α-Amino Amide Derivatives as Potential Histone Deacetylase 6 Inhibitors.

Hydroxamate, as a zinc-binding group (ZBG), prevails in the design of histone deacetylase 6(HDAC6) inhibitors due to its remarkable zinc-chelating capability. However, hydroxamate-associated genotoxicity and mutagenicity have limited the widespread application of corresponding HDAC6 inhibitors in the treatment of human diseases. To avoid such side effects, researchers are searching for novel ZBGs that may be used for the synthesis of HDAC6 inhibitors. In this study, a series of stereoisomeric compounds were designed and synthesized to discover non-hydroxamate HDAC6 inhibitors using α-amino amide as zinc-ion-chelating groups, along with a pair of enantiomeric isomers with inverted L-shaped vertical structure as cap structures. The anti-proliferative activities were determined against HL-60, Hela, and RPMI 8226 cells, and 7a and its stereoisomer 13a exhibited excellent activities against Hela cells with IC50 = 0.31 µM and IC50 = 5.19 µM, respectively. Interestingly, there is a significant difference between the two stereoisomers. Moreover, an evaluation of cytotoxicity toward human normal liver cells HL-7702 indicated its safety for normal cells. X-ray single crystal diffraction was employed to increase insights into molecule structure and activities. It was found that the carbonyl of the amide bond is on the different side from the amino and pyridine nitrogen atoms. To identify possible protein targets to clarify the mechanism of action and biological activity of 7a, a small-scale virtual screen using reverse docking for HDAC isoforms (1–10) was performed and the results showed that HDAC6 was the best receptor for 7a, suggesting that HDAC6 may be a potential target for 7a. The interaction pattern analysis showed that the α-amino amide moiety of 7a coordinated with the zinc ion of HDAC6 in a bidentate chelate manner, which is similar to the chelation pattern of hydroxamic acid. Finally, the molecular dynamics simulation approaches were used to assess the docked complex’s conformational stability. In this work, we identified 7a as a potential HDAC6 inhibitor and provide some references for the discovery of non-hydroxamic acid HDAC6 inhibitors.

Templated folding of the RTX domain of the bacterial toxin adenylate cyclase revealed by single molecule force spectroscopy

The RTX (repeats-in-toxin) domain of the bacterial toxin adenylate cyclase (CyaA) contains five RTX blocks (RTX-i to RTX-v) and its folding is essential for CyaA’s functions. It was shown that the C-terminal capping structure of RTX-v is critical for the whole RTX to fold. However, it is unknown how the folding signal transmits within the RTX domain. Here we use optical tweezers to investigate the interplay between the folding of RTX-iv and RTX-v. Our results show that RTX-iv alone is disordered, but folds into a Ca2+-loaded-β-roll structure in the presence of a folded RTX-v. Folding trajectories of RTX-iv-v reveal that the folding of RTX-iv is strictly conditional upon the folding of RTX-v, suggesting that the folding of RTX-iv is templated by RTX-v. This templating effect allows RTX-iv to fold rapidly, and provides significant mutual stabilization. Our study reveals a possible mechanism for transmitting the folding signal within the RTX domain.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3’ UTR regions

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5’ cap structure and the 3’ poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3’ untranslated region (3’ UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3’ UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3’ UTR of CLB6 mRNA.

Targeting Translational Control in Cancer Using Small Molecule Activators of PP2A

BackgroundCancer cells rely on hyperactive protein synthesis to reprogram the proteome to sustain the transformed phenotype. Thus, aberrant protein translation has emerged as a promising therapeutic target. Cap‐dependent translation is an exquisitely regulated process that requires the assembly of the eIF4F translation initiation complex. The rate limiting component of the eIF4F complex is eIF4E, which binds to the 5’ cap structure of mRNAs to initiate translation. Cap‐dependent translation is kept in check by the translation repressor 4E‐BP1; in its active hypophosphorylated form, 4E‐BP1 sequesters eIF4E to prevent translation initiation. Cancer cells use several mechanisms to bypass 4E‐BP1 translational suppres­sion, including hyperactivation of mTORC1 for hyperphos­phorylation and inactivation of 4E‐BP1; inactivation of the 4E‐BP1 phosphatase, PP2A; and profound suppression of 4E‐BP1 expression. We have found that the expression and activity of 4E‐BP1 can be restored in tumor cells using novel small molecule acti­vators of PP2A (SMAPs), a class of compounds that activate selected PP2A tumor suppressors and are being ac­tively developed as anticancer agents (Leonard et al. Cell, 2020). The objective of this study was to investigate the mechanism(s) by which SMAPs lead to restoration of the expression and activity of 4E‐BP1 in cancer cells for inhibition of hyperactive protein translation and tumor suppression.HypothesisBased on our finding that SMAPs promote PP2A‐dependent transcriptional activation of the 4E‐BP1 gene, we hypothesize that PP2A activation modulates the levels and/or activity of key transcription factors that regulate 4E‐BP1 gene expression.Results/DataWe have found that SMAPs (e.g., lead compound DT‐061) upregulate 4E‐BP1 mRNA and protein in tumor cells by inducing transcription of the 4E‐BP1 gene. Importantly, SMAPs promote the accumulation of active 4E‐BP1, capable of suppressing cap‐dependent translation in cancer cells. SMAPs promote PP2A‐dependent loss of Snail/Slug and upregulation of ATF4, transcription factors that have been implicated in negative and posi­tive control of 4E‐BP1 gene ex­pression, respectively. Snail downregulation was prevented by proteasome inhibition, indicating that Snail loss is due to proteasomal degradation. Use of GSK‐3β and PKD1 inhibitors excluded canonical mechanisms of Snail degradation in the effects of SMAPs. siRNA knockdown experiments are underway to explore the role of changes in Snail and ATF4 in SMAP‐induced upregulation of 4E‐BP1. Ongoing studies are also examining the mechanisms of SMAP/PP2A regulation of Snail/Slug and ATF4 in tumor cells.ConclusionsSMAPs promote PP2A‐dependent proteasomal degradation of Snail/Slug protein via a non‐canonical mechanism, as well as upregulation of ATF4. We propose that these effects relieve transcriptional repression of 4E‐BP1 by Snail/Slug while promoting 4E‐BP1 transcription by ATF4, leading to a robust increase in 4E‐BP1 levels. 4E‐BP1 translation repressive activity is ensured by PP2A‐induced 4E‐BP1 dephosphorylation. Thus, SMAPs offer a powerful tool for therapeutic targeting of aberrant protein translation in cancer.

TGS1 mediates 2,2,7-trimethyl guanosine capping of the human telomerase RNA to direct telomerase dependent telomere maintenance

Pathways that direct the selection of the telomerase-dependent or recombination-based, alternative lengthening of telomere (ALT) maintenance pathway in cancer cells are poorly understood. Using human lung cancer cells and tumor organoids we show that formation of the 2,2,7-trimethylguanosine (TMG) cap structure at the human telomerase RNA 5′ end by the Trimethylguanosine Synthase 1 (TGS1) is central for recruiting telomerase to telomeres and engaging Cajal bodies in telomere maintenance. TGS1 depletion or inhibition by the natural nucleoside sinefungin impairs telomerase recruitment to telomeres leading to Exonuclease 1 mediated generation of telomere 3′ end protrusions that engage in RAD51-dependent, homology directed recombination and the activation of key features of the ALT pathway. This indicates a critical role for 2,2,7-TMG capping of the RNA component of human telomerase (hTR) in enforcing telomerase-dependent telomere maintenance to restrict the formation of telomeric substrates conductive to ALT. Our work introduces a targetable pathway of telomere maintenance that holds relevance for telomere-related diseases such as cancer and aging.

Recent Advances in Modified Cap Analogs: Synthesis, Biochemical Properties, and mRNA Based Vaccines.

The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7G[5′]ppp[5′]N (where N=G, A, C or U), present at the 5′‐end of many eukaryotic cellular and viral RNAs and several non‐coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.

Oxidative Damage Induces a Vacancy G-Quadruplex That Binds Guanine Metabolites: Solution Structure of a cGMP Fill-in Vacancy G-Quadruplex in the Oxidized BLM Gene Promoter.

Guanine (G)-oxidation to 8-oxo-7,8-dihydroguanine (OG) by reactive oxygen species in genomic DNA has been implicated with various human diseases. G-quadruplex (G4)-forming sequences in gene promoters are highly susceptible to G-oxidation, which can subsequently cause gene activation. However, the underlying G4 structural changes that result from OG modifications remain poorly understood. Herein, we investigate the effect of G-oxidation on the BLM gene promoter G4. For the first time, we show that OG can induce a G-vacancy-containing G4 (vG4), which can be filled in and stabilized by guanine metabolites and derivatives. We determined the NMR solution structure of the cGMP-fill-in oxidized BLM promoter vG4. This is the first complex structure of an OG-induced vG4 from a human gene promoter sequence with a filled-in guanine metabolite. The high-resolution structure elucidates the structural features of the specific 5'-end cGMP-fill-in for the OG-induced vG4. Interestingly, the OG is removed from the G-core and becomes part of the 3'-end capping structure. A series of guanine metabolites and derivatives are evaluated for fill-in activity to the oxidation-induced vG4. Significantly, cellular guanine metabolites, such as cGMP and GTP, can bind and stabilize the OG-induced vG4, suggesting their potential regulatory role in response to oxidative damage in physiological and pathological processes. Our work thus provides exciting insights into how oxidative damage and cellular metabolites may work together through a G4-based epigenetic feature for gene regulation. Furthermore, the NMR structure can guide the rational design of small-molecule inhibitors that specifically target the oxidation-induced vG4s.

Synthesis of Doped/Hybrid Carbon Dots and Their Biomedical Application.

Carbon dots (CDs) are a novel type of carbon-based nanomaterial that has gained considerable attention for their unique optical properties, including tunable fluorescence, stability against photobleaching and photoblinking, and strong fluorescence, which is attributed to a large number of organic functional groups (amino groups, hydroxyl, ketonic, ester, and carboxyl groups, etc.). In addition, they also demonstrate high stability and electron mobility. This article reviews the topic of doped CDs with organic and inorganic atoms and molecules. Such doping leads to their functionalization to obtain desired physical and chemical properties for biomedical applications. We have mainly highlighted modification techniques, including doping, polymer capping, surface functionalization, nanocomposite and core-shell structures, which are aimed at their applications to the biomedical field, such as bioimaging, bio-sensor applications, neuron tissue engineering, drug delivery and cancer therapy. Finally, we discuss the key challenges to be addressed, the future directions of research, and the possibilities of a complete hybrid format of CD-based materials.

CMTR1 is recruited to transcription start sites and promotes ribosomal protein and histone gene expression in embryonic stem cells.

CMTR1 (cap methyltransferase 1) catalyses methylation of the first transcribed nucleotide of RNAPII transcripts (N1 2′-O-Me), creating part of the mammalian RNA cap structure. In addition to marking RNA as self, N1 2′-O-Me has ill-defined roles in RNA expression and translation. Here, we investigated the gene specificity of CMTR1 and its impact on RNA expression in embryonic stem cells. Using chromatin immunoprecipitation, CMTR1 was found to bind to transcription start sites (TSS) correlating with RNAPII levels, predominantly binding at histone genes and ribosomal protein (RP) genes. Repression of CMTR1 expression resulted in repression of RNAPII binding at the TSS and repression of RNA expression, particularly of histone and RP genes. In correlation with regulation of histones and RP genes, CMTR1 repression resulted in repression of translation and induction of DNA replication stress and damage. Indicating a direct role for CMTR1 in transcription, addition of recombinant CMTR1 to purified nuclei increased transcription of the histone and RP genes. CMTR1 was found to be upregulated during neural differentiation and there was an enhanced requirement for CMTR1 for gene expression and proliferation during this process. We highlight the distinct roles of the cap methyltransferases RNMT and CMTR1 in target gene expression and differentiation.

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing.

The mRNA 5' cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-containing genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

Reversal of G-Quadruplexes' Role in Translation Control When Present in the Context of an IRES.

G-quadruplexes (GQs) are secondary nucleic acid structures that play regulatory roles in various cellular processes. G-quadruplex-forming sequences present within the 5′ UTR of mRNAs can function not only as repressors of translation but also as elements required for optimum function. Based upon previous reports, the majority of the 5′ UTR GQ structures inhibit translation, presumably by blocking the ribosome scanning process that is essential for detection of the initiation codon. However, there are certain mRNAs containing GQs that have been identified as positive regulators of translation, as they are needed for translation initiation. While most cellular mRNAs utilize the 5′ cap structure to undergo cap-dependent translation initiation, many rely on cap-independent translation under certain conditions in which the cap-dependent initiation mechanism is not viable or slowed down, for example, during development, under stress and in many diseases. Cap-independent translation mainly occurs via Internal Ribosomal Entry Sites (IRESs) that are located in the 5′ UTR of mRNAs and are equipped with structural features that can recruit the ribosome or other factors to initiate translation without the need for a 5′ cap. In this review, we will focus only on the role of RNA GQs present in the 5′ UTR of mRNAs, where they play a critical role in translation initiation, and discuss the potential mechanism of this phenomenon, which is yet to be fully delineated.