Biology subjects

Cramer, P.

Publications and source records attributed to Cramer, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Unique transcriptomes define naive, primed and paused pluripotent states in mouse embryonic stem cells. Here we perform transient transcriptome sequencing (TT-seq) to de novo define and quantify coding and non-coding transcription units (TUs) in different pluripotent states. We observe a global reduction of RNA synthesis, total RNA amount and turnover rates in ground state naive cells (2i) and paused pluripotency (mTORi). We demonstrate that elongation velocity can be reliably estimated from TT-seq nascent RNA and RNA polymerase II occupancy and observe a transcriptome-wide attenuation of elongation velocity in the two inhibitor-induced states. We also discover a relationship between elongation velocity and termination read-through distance. Our analysis suggests that steady-state transcriptomes in mouse ES cells are controlled predominantly on the level of RNA synthesis, and that signaling pathways governing different pluripotent states immediately control key parameters of transcription.

systems biology↗

bioRxiv2021

Mechanism of molnupiravir-induced SARS-CoV-2 mutagenesis

Molnupiravir is an orally available antiviral drug candidate that is in phase III trials for the treatment of COVID-19 patients1,2. Molnupiravir increases the frequency of viral RNA mutations3,4 and impairs SARS-CoV-2 replication in animal models4-6 and in patients2. Here we establish the molecular mechanisms that underlie molnupiravir-induced RNA mutagenesis by the RNA-dependent RNA polymerase (RdRp) of the coronavirus SARS-CoV-2. Biochemical assays show that the RdRp readily uses the active form of molnupiravir, {beta}-D-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of CTP or UTP. Incorporation of NHC monophosphate into nascent RNA does not impair further RdRp progression. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp-RNA complexes containing mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism likely applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir.

biochemistry↗

bioRxiv2021

Scaling of cellular proteome with ploidy

Ploidy changes are frequent in nature and contribute to evolution, functional specialization and tumorigenesis (1,2). Analysis of model organisms of different ploidies revealed that increased ploidy leads to an increase in cell and nuclear volume, reduced proliferation (2-4), metabolic changes (5), lower fitness (6,7), and increased genomic instability (8,9), but the underlying mechanisms remain poorly understood. To investigate how the gene expression changes with cellular ploidy, we analyzed isogenic series of budding yeasts from 1N to 4N. We show that mRNA and protein abundance scales allometrically with ploidy, with tetraploid cells showing only threefold increase in proteins compared to haploids. This ploidy-specific scaling occurs via decreased rRNA and ribosomal protein abundance and reduced translation. We demonstrate that the Tor1 activity is reduced with increasing ploidy, which leads to rRNA gene repression via a novel Tor1-Sch9-Tup1 signaling pathway. mTORC1 and S6K activity are also reduced in human tetraploid cells and the concomitant increase of the Tup1 homolog Tle1 downregulates the rDNA transcription. Our results revealed a novel conserved mTORC1-S6K-Tup1/Tle1 pathway that ensures proteome remodeling in response to increased ploidy.

cell biology↗

bioRxiv2021

Dimeric form of SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Structures of the RdRp revealed a monomeric enzyme composed of the catalytic subunit nsp12, two copies of subunit nsp8, and one copy of subunit nsp7. Here we report an alternative, dimeric form of the coronavirus polymerase and resolve its structure at 4.8 [A] resolution. In this structure, the two RdRps contain only one copy of nsp8 and dimerize via their nsp7 subunits to adopt an antiparallel arrangement. We speculate that the RdRp dimer facilitates template switching during production of sub-genomic RNAs for transcription.

molecular biology↗

bioRxiv2021

Sequence determinants of human gene regulatory elements

DNA determines where and when genes are expressed, but the full set of sequence determinants that control gene expression is not known. To obtain a global and unbiased view of the relative importance of different sequence determinants in gene expression, we measured transcriptional activity of DNA sequences that are in aggregate [~]100 times longer than the human genome in three different cell types. We show that enhancers can be classified to three main types: classical enhancers1, closed chromatin enhancers and chromatin-dependent enhancers, which act via different mechanisms and differ in motif content. Transcription factors (TFs) act generally in an additive manner with weak grammar, with classical enhancers increasing expression from promoters by a mechanism that does not involve specific TF-TF interactions. Few TFs are strongly active in a cell, with most activities similar between cell types. Chromatin-dependent enhancers are enriched in forkhead motifs, whereas classical enhancers contain motifs for TFs with strong transactivator domains such as ETS and bZIP; these motifs are also found at transcription start site (TSS)-proximal positions. However, some TFs, such as NRF1 only activate transcription when placed close to the TSS, and others such as YY1 display positional preference with respect to the TSS. TFs can thus be classified into four non-exclusive subtypes based on their transcriptional activity: chromatin opening, enhancing, promoting and TSS determining factors - consistent with the view that the binding motif is the only atomic unit of gene expression.

genomics↗

bioRxiv2021

Structure of human Mediator-RNA polymerase II transcription pre-initiation complex

Mediator is a conserved coactivator that enables regulated transcription initiation from eukaryotic protein-coding genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate RNA polymerase II (Pol II) phosphorylation and promoter escape1-6. Here we prepare a 20-subunit recombinant human Mediator, reconstitute a 50-subunit Mediator-PIC complex, and resolve the complex structure by cryo-EM at an overall resolution of 4.5 [A]. Mediator binds with its head module to the Pol II stalk and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions in the corresponding yeast complex7-9. One end of Mediator contains the metazoan-specific subunits MED27-MED30, which associate with exposed regions in MED14 and MED17 to form the proximal part of the tail module that binds activators. The opposite end of Mediator positions the flexibly linked CDK-activating kinase (CAK) of the general transcription factor TFIIH near the C-terminal repeat domain (CTD) of Pol II. The Mediator shoulder domain holds the CAK subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of CDK7 to allosterically stimulate CTD phosphorylation and Pol II escape from the promoter.

molecular biology↗

bioRxiv2021

The total mRNA concentration buffering system in yeast is global rather than gene-specific

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentrations and their synthesis rates in non sense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one.

molecular biology↗

bioRxiv2020

Structural basis of nucleosome transcription mediated by Chd1 and FACT

Transcription of eukaryotic protein-coding genes requires passage of RNA polymerase II (Pol II) through nucleosomes. Efficient Pol II passage through nucleosomes depends on the chromatin remodelling factor Chd11 and the histone chaperone FACT2. How Chd1 and FACT mediate Pol II passage through nucleosomes remains unclear. Here we first show that Chd1 and FACT cooperate with the elongation factors Spt4/5 and TFIIS to facilitate Pol II transcription through a nucleosome in a defined biochemical system. We then determine cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT at 2.9 [A] and 3.1 [A] resolution, respectively. In the first structure, transcribing Pol II has partially unwrapped nucleosomal DNA and exposed the proximal histone H2A/H2B dimer, which is bound by the acidic N-terminal region of Spt5 (Spt5N). The inhibitory DNA-binding region of Chd1 is released3 and the Chd1 translocase adopts an activated state that is poised to pump DNA towards Pol II. In the second structure, transcribing Pol II has generated a partially unravelled nucleosome that binds FACT in a manner that excludes Chd1 and Spt5N. These results suggest a dynamic model of Pol II passage through a nucleosome. In the model, Pol II enters the nucleosome4, activates Chd1 by releasing its DNA-binding region, and thereby stimulates its own progression. Pol II progression then enables FACT binding, liberates Chd1 and Spt5N, and eventually displaces a complex of FACT with histones that is transferred to upstream DNA.

biochemistry↗

bioRxiv2020

Mechanism of SARS-CoV-2 polymerase inhibition by remdesivir

Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients1-4. The active form of remdesivir acts as a nucleoside analogue and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-25-7. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls6,8. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3-nucleotide of the RNA product is matched with the template base, and this may prevent proofreading by the viral 3-exonuclease that recognizes mismatches9,10. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

biochemistry↗

bioRxiv2020

Structure of a transcribing RNA polymerase II-U1 snRNP complex

To initiate co-transcriptional splicing, RNA polymerase II (Pol II) recruits U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent pre-mRNA. Here we report the cryo-EM structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the 5 splice site in pre-mRNA near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5 splice site, leading to formation of an intron loop. Loop formation may facilitate scanning of the nascent pre-mRNA for the 3 splice site and enable prespliceosome assembly and functional pairing of distant intron ends. Our results provide a starting point for a mechanistic analysis of co-transcriptional splicing and the biogenesis of mRNA isoforms during alternative splicing.

molecular biology↗

bioRxiv2020

Integrator is a genome-wide attenuator of non-productive transcription

Termination of RNA polymerase II (RNAPII) transcription in metazoans relies largely on the Cleavage and Polyadenylation (CPA) and Integrator (INT) complexes originally found to act at the ends of protein-coding and snRNA genes, respectively. Here we monitor CPA- and INT-dependent termination activities genome-wide, including over 8000 previously unannotated transcription units (TUs), that produce unstable RNA. We verify the global activity of CPA, that occurs at pA sites indiscriminately of their positioning relative to the TU promoter. We also identify a global activity of INT, which is, however, largely sequence-independent and restricted to a ~3 kb promoter-proximal region. Our analyses suggest two functions of genome-wide INT activity; it dampens transcriptional output from weak promoters and it provides quality-control of RNAPII complexes, that are unfavorably configured for transcriptional elongation. We suggest that the function of INT in stable snRNA production is an exception from its general cellular role, attenuation of non-productive transcription.

cell biology↗

bioRxiv2020

Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.7 A inside cells

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 [A]. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.

biophysics↗

bioRxiv2020

Structure of replicating SARS-CoV-2 polymerase

The coronavirus SARS-CoV-2 uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. Here we present the cryo-electron microscopic structure of the SARS-CoV-2 RdRp in its replicating form. The structure comprises the viral proteins nsp12, nsp8, and nsp7, and over two turns of RNA template-product duplex. The active site cleft of nsp12 binds the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the RNA duplex as it exits. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged sliding poles that may enable processive replication of the long coronavirus genome. Our results will allow for a detailed analysis of the inhibitory mechanisms used by antivirals such as remdesivir, which is currently in clinical trials for the treatment of coronavirus disease 2019 (COVID-19).

biophysics↗

bioRxiv2020

Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Arrays of regularly spaced nucleosomes dominate chromatin and are often phased by alignment to reference sites like active promoters. How the distances between nucleosomes (spacing), and between phasing sites and nucleosomes are determined remains unclear, and specifically, how ATP dependent chromatin remodelers impact these features. Here, we used genome-wide reconstitution to probe how Saccharomyces cerevisiae ATP dependent remodelers generate phased arrays of regularly spaced nucleosomes. We find that remodelers bear a functional element named the ruler that determines spacing and phasing in a remodeler-specific way. We use structure-based mutagenesis to identify and tune the ruler element residing in the Nhp10 and Arp8 modules of the INO80 remodeler complex. Generally, we propose that a remodeler ruler regulates nucleosome sliding direction bias in response to (epi)genetic information. This finally conceptualizes how remodeler-mediated nucleosome dynamics determine stable steady-state nucleosome positioning relative to other nucleosomes, DNA bound factors, DNA ends and DNA sequence elements.

biochemistry↗

bioRxiv2020

In-cell architecture of an actively transcribing-translating expressome

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.

molecular biology↗

bioRxiv2019

Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome

Chromatin remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted regions and transcriptionally active promoters in the eukaryote genome. The structure of the Saccharomyces cerevisiae SWI/SNF family member RSC in complex with a nucleosome substrate reveals five protein modules and suggests key features of the remodelling mechanism. A DNA-interacting module grasps extra-nucleosomal DNA and helps to recruit RSC to promoters. The ATPase and arm modules sandwich the nucleosome disc with their SnAC and finger elements, respectively. The translocase motor engages with the edge of the nucleosome at superhelical location +2 to pump DNA along the nucleosome, resulting in a sliding of the histone octamer along DNA. The results elucidate how nucleosome-depleted regions are formed and provide a basis for understanding human chromatin remodelling complexes of the SWI/SNF family and the consequences of cancer mutations that frequently occur in these complexes.

molecular biology↗

bioRxiv2019

Nucleosome-CHD4 chromatin remodeller structure explains human disease mutations

Chromatin remodelling plays important roles in gene regulation during development, differentiation and in disease. The chromatin remodelling enzyme CHD4 is a component of the NuRD and ChAHP complexes that are involved in gene repression. Here we report the cryo-electron microscopy (cryo-EM) structure of Homo sapiens CHD4 engaged with a nucleosome core particle in the presence of the non-hydrolysable ATP analogue AMP-PNP at an overall resolution of 3.1 [A]. The ATPase motor of CHD4 binds and distorts nucleosomal DNA at super-helical location (SHL) +2, supporting the twist defect model of chromatin remodelling. CHD4 does not induce unwrapping of terminal DNA, in contrast to its homologue Chd1, which functions in gene activation. Our results also rationalize the effect of CHD4 mutations that are associated with cancer or the intellectual disability disorder Sifrim-Hitz-Weiss syndrome.

molecular biology↗

bioRxiv2019

Transient transcriptome sequencing: computational pipeline to quantify genome-wide RNA kinetic parameters and transcriptional enhancer activity

In the accompanying chapter (Gressel, Lidschreiber, Cramer), we describe the detailed experimental protocol for transient transcriptome sequencing (TT-seq). TT-seq detects metabolically labeled, newly synthesized RNA fragments genome-wide in living cells. TT-seq can monitor gene activity and the dynamics of enhancer landscapes with great sensitivity, but this requires careful computational analysis of the data. In this manuscript, we present the bioinformatics workflow used to analyze TT-seq data. In particular, we describe pre-processing steps, including a reliable and robust normalization strategy, and several downstream analysis tools that enable the user to quantify RNA synthesis, splicing and degradation activities. Together, these tools form a comprehensive analysis pipeline that can be adapted to almost any TT-seq application.

molecular biology↗