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Land of wondrous cold : the race to discover Antarctica and unlock the secrets of its ice
\"A history of the first race to Antarctica that weaves the great polar discoveries of the nineteenth century with scientific breakthroughs of the modern era. Antarctica, the ice kingdom hosting the South Pole, looms large in the human imagination. The secrets of this vast frozen desert have long tempted explorers, but its brutal climate and glacial shores notoriously resist human intrusion. Land of Wondrous Cold tells a gripping story of the pioneer nineteenth-century voyages, when British, French, and American commanders raced to penetrate Antarctica's glacial rim for unknown lands beyond. These intrepid Victorian explorers-James Ross, Dumont D'Urville, and Charles Wilkes-laid the foundation for our current understanding of Terra Australis Incognita. Today, the white continent poses new challenges, as scientists race to uncover Earth's climate history recorded in the south polar ice and ocean floor, and to monitor the increasing instability of the Antarctic ice cap, which threatens inundation of coastal cities worldwide. Interweaving the breakthrough research of the modern Ocean Drilling Program with the dramatic discovery tales of their Victorian-era forerunners, Gillen D'Arcy Wood describes Antarctica's role in a planetary drama of plate tectonics, climate change, and species evolution stretching back more than thirty million years. An original, multifaceted portrait of the polar continent emerges, illuminating our profound connection to Antarctica in its past, present, and future incarnations. A deep-time history of monumental scale, Land of Wondrous Cold brings the remotest of worlds within close reach-an Antarctica vital to both planetary history and human fortunes\"-- Provided by publisher.
Book
mRNA ageing shapes the Cap2 methylome in mammalian mRNA
The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2′-
O
-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively
1
,
2
. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.
Cap2 methylation increases on transcripts as they age, reducing activation of innate immunity.
Journal Article
Photoactivatable mRNA 5′ Cap Analogs for RNA‐Protein Crosslinking
Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA‐based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5′‐end, called 5′‐cap. Depending on the 5′‐terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5′‐end variants. Here, a series of 8 new synthetic 5′‐cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap‐protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap‐binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap‐binding subunit in a multi‐protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA‐related processes. A deeper understanding of mRNA translation mechanisms requires new molecular tools to identify binders of different mRNA 5′‐end variants. A series of trinucleotide 5′‐cap analogs that allow for the straightforward preparation of RNA molecules with photoreactive tags capable of forming specific crosslinks with model cap‐binding proteins are reported. Such RNAs have also proved useful for identifying the cap‐binding subunit in multi‐protein complexes.
Journal Article
5′-Cap sequestration is an essential determinant of HIV-1 genome packaging
HIV-1 selectively packages two copies of its 5′-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5′ leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5′ leader. ΨCES lacks a 5′-tandem hairpin element that sequesters the 5′ cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5′ ribozyme to ΨCES to enable cotranscriptional shedding of the 5′ cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5′ cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5′-capped RNA genomes.
Journal Article
Discovery of m(7)G-cap in eukaryotic mRNAs
Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2'-O-methylated nucleotide in the 5' end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5' terminal structure (referred later to as \"cap\" or \"m(7)G-cap\") in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5'-cap m(7)GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5' cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping.
Journal Article
eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation
The initiation protein eIF3d serves as an alternative cap-recognition factor for a subclass of mRNAs, such as c-Jun; the high-resolution structure of the eIF3d cap-binding domain can be modelled onto the cap structure, defining interactions that are needed for translation of these mRNAs.
An alternative 5′ mRNA capping pathway
Recruitment of the ribosome to eukaryotic messenger RNAs (mRNAs) involves interaction between the translation initiation factor, eIF4E, and a specialized nucleotide, or 'cap', at the 5′ end of the mRNA. eIF4E is inactivated under certain conditions, but translation of numerous mRNAs is maintained. Jamie Cate and colleagues describe how another initiation protein, eIF3d, serves as an alternative cap-recognition factor for a subclass of mRNAs, such as
c-Jun
. The high-resolution structure of the eIF3d cap-binding domain can be modelled onto the cap structure, defining interactions that are needed for translation of these mRNAs. They also find that
c-Jun
mRNA contains an element that inhibits eIF4E recruitment.
Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression
1
despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs
2
such as the cell proliferation regulator
c-Jun
(also known as
JUN
). The
c-Jun
mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.
Journal Article
2′-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography
The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5′-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2′-O position of the first nucleotide. To investigate the conformational changes of the complex during 2′-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2′-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.
Journal Article
mRNA as novel technology for passive immunotherapy
While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.
Journal Article
Structural insight into cap-snatching and RNA synthesis by influenza polymerase
Influenza virus polymerase uses a capped primer, derived by ‘cap-snatching’ from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves
in situ
rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.
Atomic resolution crystal structures of influenza A and B polymerases are presented; comparison of these structures provides mechanistic insight into influenza polymerase functions, explaining the processes of cap-snatching and cap-dependent priming, which are unique to segmented negative-strand RNA viruses.
Complete structure of influenza polymerase A
Stephen Cusack and colleagues have solved the crystal structure of the complete influenza polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. In the first of two papers they present the structure of the polymerase from a bat-specific influenza A virus, which is evolutionarily close to human/avian influenza A strains. The second paper presents the structure of the polymerase from a human isolate of influenza B. Together, the structures provide a wealth of information about how the influenza polymerase functions and how the different subunits interact with each other.
Journal Article