Biology subjects

Oerum, S.

Publications and source records attributed to Oerum, S..

5 recordsLinked to original sources

bioRxiv2023

Structural basis for human mitochondrial tRNA maturation

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), mt-RNase P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for a tRNA methyltransferase complex in mt-RNase P cleavage activity is not fully understood. Moreover, the mechanisms of pre-tRNA 3-cleavage and CCA addition have also yet to be uncovered. Here we report cryo-EM structures that visualise these events, revealing the molecular basis for the first steps of RNA-transcript processing in human mitochondria. Altogether, the structures visualise four steps of mitochondrial tRNA maturation: 5 and 3 tRNA-end processing, methylation and 3-CCA addition, and explain the defined sequential order of tRNA processing. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA processing and 3-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-variable mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes.

biochemistry↗

bioRxiv2020

Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3 UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.

molecular biology↗

bioRxiv2020

A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR

The SARS-CoV-2 pandemic has created an urgent need for large amounts of diagnostic tests to detect viral RNA, which commercial suppliers are increasingly unable to deliver. In addition to the lack of availability, the current methods do not always fully inactivate the virus. Together, this calls for the development of safer methods for extraction and detection of viral RNA from patient samples that utilise readily available reagents and equipment present in most standard laboratories. We present a rapid and straightforward RNA extraction protocol for inactivating the SARS-CoV-2 virus that uses standard lab reagents. This protocol expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method circumvents the need for commercial RNA purification kits, takes about 30 minutes from swab to PCR-ready viral RNA, and enables downstream detection of SARS-CoV-2 by RT-qPCR with very high sensitivity (~4 viral RNA copies per RT-qPCR). In summary, we present a rapid, safe and sensitive method for high-throughput detection of SARS-CoV-2, that can be conducted in any laboratory equipped with a qPCR machine.

microbiology↗

bioRxiv2019

Electrospray Surface Charge Describes Protein Molecular Motion

The electrospray mass/charge distribution for a protein is an instantaneous measurement of surface areas during the transition from liquid to gas phase. Protonation is dependent upon surface area and surface area is related to protein folding under all conditions. M/z distributions for proteins and protein analogues all with different degrees of entropy were compared. Rigid dipeptide self-assemblies of any size possess a single m/z, whereas protein always displays multiple m/z distributions. Native proteins have narrow, defined m/z distributions, while denatured proteins and synthetic homopolypetides possess the widest possible m/z distributions. These observations are consistent with dynamic changes in surface area resulting from molecular motion.

biophysics↗

bioRxiv2017

A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency

Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations.\n\nAuthor SummaryPerrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.

genetics↗