Biology subjects

Jenni, S.

Publications and source records attributed to Jenni, S..

5 recordsLinked to original sources

bioRxiv2019

Single cell tri-channel-processing reveals structural variation landscapes and complex rearrangement processes

Structural variation (SV), where rearrangements delete, duplicate, invert or translocate DNA segments, is a major source of somatic cell variation. It can arise in rapid bursts, mediate genetic heterogenity, and dysregulate cancer-related pathways. The challenge to systematically discover SVs in single cells remains unsolved, with copy-neutral and complex variants typically escaping detection. We developed single cell tri-channel-processing (scTRIP), a computational framework that jointly integrates read depth, template strand and haplotype phase to comprehensively discover SVs in single cells. We surveyed SV landscapes of 565 single cell genomes, including transformed epithelial cells and patient-derived leukemic samples, and discovered abundant SV classes including inversions, translocations and large-scale genomic rearrangements mediating oncogenic dysregulation. We dissected the molecular karyotype of the leukemic samples and examined their clonal structure. Different from prior methods, scTRIP also enabled direct detection and discrimination of SV mutational processes in individual cells, including breakage-fusion-bridge cycles. scTRIP will facilitate studies of clonal evolution, genetic mosaicism and somatic SV formation, and could improve disease classification for precision medicine.

genomics↗

bioRxiv2019

Structure of a rabies virus polymerase complex from electron cryo-microscopy

Non-segmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS-RNA viruses carry all the enzymatic functions required for viral mRNA transcription and replication: RNA polymerization, mRNA capping, cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 [A] resolution. The complex closely resembles vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a pre-initiation conformation. Comparison with the likely elongation state, seen in two partial structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.

molecular biology↗

bioRxiv2019

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated and polyadenylated mRNAs and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates function and conformation of the L protein. We report the structure of vesicular stomatitis virus L in complex with its P cofactor determined by electron cryomicroscopy at 3.0 [A] resolution, enabling us to visualize bound segments of P. The contacts of three P segments with multiple L domains show how P induces a closed, compact, initiation-competent conformation. Binding of P to L positions its N-terminal domain adjacent to a putative RNA exit channel for efficient encapsidation of newly synthesized genomes with the nucleoprotein and orients its C-terminal domain to interact with the RNP template. The model shows that a conserved tryptophan in the priming loop can support the initiating 5-nucleotide.

molecular biology↗

bioRxiv2019

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

biophysics↗

bioRxiv2019

In situ structure of rotavirus VP1 RNA-dependent RNA polymerase

Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the \"double-layer particle\" or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each fivefold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual fivefold positions. We have analyzed intact virions (\"triple-layer particles\" or TLPs), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg2+, and S-adenosyl methionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, dsRNA genome.

molecular biology↗