Biology subjects

Hillen, H. S.

Publications and source records attributed to Hillen, H. S..

8 recordsLinked to original sources

bioRxiv2026

Structure of the human mitochondrial RNA degradosomereveals a distinct mode of helicase-nuclease coupling

RNA degradation shapes cellular and organellar gene expression. In human mitochondria, this process is mediated by a dedicated degradosome comprising the helicase SUV3 and the exoribonuclease PNPase, but how these enzymes assemble and cooperate to degrade structured RNA has remained unknown. Here, we report the cryo-EM structure of the complete human mitochondrial RNA degradosome bound to RNA. The structure reveals an asymmetric heteropentamer composed of a SUV3 dimer and the trimeric PNPase. Degradosome assembly is accompanied by conformational rearrangements in PNPase that reshape the RNA-entry channel and generate an asymmetric S1-domain platform for SUV3 binding. This creates a continuous electropositive path from SUV3 to PNPase, suggesting how RNA may be guided during degradation. Together, these findings reveal a mode of helicase-nuclease coupling distinct from other RNA degradation machineries and provide a structural framework for understanding human mitochondrial RNA degradation, its regulation, and association with disease.

biochemistry↗

bioRxiv2024

Structural basis of Nipah virus replication

Nipah virus (NiV) is a non-segmented negative-strand RNA virus (nsNSV) with high pandemic potential, as it frequently causes zoonotic outbreaks and can be transmitted from human to human. Its RNA-dependent RNA polymerase (RdRp) complex carries out viral genome replication and transcription and is therefore an attractive drug target. However, to date no structural data is available on the NiV RdRp complex. Here, we report cryo-EM structures of NiV RdRp in the apo and in an early elongation state with RNA and incoming substrate bound. The structure of the apo enzyme reveals the architecture of the NiV RdRp complex, which shows a high degree of similarity to other nsNSV RdRps. The structure of the RNA-bound NiV RdRp shows how the enzyme interacts with template and product RNA during early replication and how nucleoside triphosphates are bound in the active site. Comparisons show that RNA binding leads to rearrangements of key elements in the RdRp core and to ordering of the flexible C-terminal domains of NiV L required for RNA capping. Taken together, these results reveal the first structural snapshots of an actively replicating nsNSV RdRp and provide insights into the mechanisms of genome replication and transcription by NiV and related viruses.

biochemistry↗

bioRxiv2024

FASTKD5 processes mitochondrial pre-mRNAs at non-canonical cleavage sites

The regulation of mammalian mitochondrial gene expression is largely post-transcriptional and the first step in translating the 13 polypeptides encoded in mtDNA is endonucleolytic cleavage of the primary polycistronic transcripts. As the rRNAs and most of the mRNAs in mtDNA are flanked by tRNAs, the release of the mature RNAs occurs mostly by excision of the tRNAs. Processing the non-canonical mRNAs, not flanked by tRNAs, requires FASTKD5, but the molecular mechanism remains unknown. To investigate this, we created and characterized a knockout cell line to use as an assay system. The absence of FASTKD5 resulted in a severe combined OXPHOS assembly defect due to the inability to translate mRNAs with unprocessed 5-UTRs. Analysis of RNA processing of FASTKD5 variants allowed us to map amino acid residues essential for function. Remarkably, this map was RNA substrate-specific, arguing against a one size fits all model. A reconstituted in vitro system with purified FASTKD5 protein and synthetic RNA substrates showed that FASTKD5 on its own was able to cleave client substrates correctly, but not non-specific RNA sequences. These results establish FASTKD5 as the missing piece of the biochemical machinery required to completely process the primary mitochondrial transcript.

molecular 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

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

Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

molecular biology↗

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 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↗