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Article ; Online: Organization and expression of the mammalian mitochondrial genome.

Rackham, Oliver / Filipovska, Aleksandra

2022 Volume 23, Issue 10, Page(s) 606–623

Abstract: The mitochondrial genome encodes core subunits of the respiratory chain that drives oxidative phosphorylation and is, therefore, essential for energy conversion. Advances in high-throughput sequencing technologies and cryoelectron microscopy have shed ... ...

Abstract	The mitochondrial genome encodes core subunits of the respiratory chain that drives oxidative phosphorylation and is, therefore, essential for energy conversion. Advances in high-throughput sequencing technologies and cryoelectron microscopy have shed light on the structure and organization of the mitochondrial genome and revealed unique mechanisms of mitochondrial gene regulation. New animal models of impaired mitochondrial protein synthesis have shown how the coordinated regulation of the cytoplasmic and mitochondrial translation machineries ensures the correct assembly of the respiratory chain complexes. These new technologies and disease models are providing a deeper understanding of mitochondrial genome organization and expression and of the diseases caused by impaired energy conversion, including mitochondrial, neurodegenerative, cardiovascular and metabolic diseases. They also provide avenues for the development of treatments for these conditions.
MeSH term(s)	Animals ; Cryoelectron Microscopy ; Genome, Mitochondrial ; Mammals/genetics ; Mammals/metabolism ; Mitochondria/genetics ; Mitochondrial Proteins/genetics ; Mitochondrial Proteins/metabolism ; Oxidative Phosphorylation
Chemical Substances	Mitochondrial Proteins
Language	English
Publishing date	2022-04-22
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	2035157-4
ISSN	1471-0064 ; 1471-0056
ISSN (online)	1471-0064
ISSN	1471-0056
DOI	10.1038/s41576-022-00480-x
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Article ; Online: Shackled ribosomes unleashed.

Filipovska, Aleksandra / Rackham, Oliver

Nature chemical biology

2022 Volume 18, Issue 9, Page(s) 918–920

MeSH term(s)	Ribosomes
Language	English
Publishing date	2022-07-14
Publishing country	United States
Document type	Journal Article ; Comment
ZDB-ID	2202962-X
ISSN	1552-4469 ; 1552-4450
ISSN (online)	1552-4469
ISSN	1552-4450
DOI	10.1038/s41589-022-01020-8
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Article ; Online: Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria.

Rudler, Danielle L / Siira, Stefan J / Rackham, Oliver / Filipovska, Aleksandra

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2661, Page(s) 317–328

Abstract: RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the ... ...

Abstract	RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.
MeSH term(s)	Ribonucleases/metabolism ; Ribosomes/metabolism ; RNA/chemistry ; Mitochondrial Ribosomes/metabolism ; Mitochondria/genetics ; Mitochondria/metabolism ; Endoribonucleases/metabolism ; Ribonuclease, Pancreatic/metabolism
Chemical Substances	Ribonucleases (EC 3.1.-) ; RNA (63231-63-0) ; Endoribonucleases (EC 3.1.-) ; Ribonuclease, Pancreatic (EC 3.1.27.5)
Language	English
Publishing date	2023-05-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3171-3_18
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Article ; Online: Frankenstein Cas9: engineering improved gene editing systems.

Vos, Pascal D / Filipovska, Aleksandra / Rackham, Oliver

Biochemical Society transactions

2022 Volume 50, Issue 5, Page(s) 1505–1516

Abstract: The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an ... ...

Abstract	The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.
MeSH term(s)	Gene Editing ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Endonucleases/genetics ; Endonucleases/metabolism
Chemical Substances	RNA, Guide, CRISPR-Cas Systems ; Endonucleases (EC 3.1.-)
Language	English
Publishing date	2022-10-28
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	184237-7
ISSN	1470-8752 ; 0300-5127
ISSN (online)	1470-8752
ISSN	0300-5127
DOI	10.1042/BST20220873
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Article ; Online: OmicsVolcano: software for intuitive visualization and interactive exploration of high-throughput biological data.

Kuznetsova, Irina / Lugmayr, Artur / Rackham, Oliver / Filipovska, Aleksandra

STAR protocols

2021 Volume 2, Issue 1, Page(s) 100279

Abstract: Advances in omics technologies have generated exponentially larger volumes of biological data; however, their analyses and interpretation are limited to computationally proficient scientists. We created OmicsVolcano, an interactive open-source software ... ...

Abstract	Advances in omics technologies have generated exponentially larger volumes of biological data; however, their analyses and interpretation are limited to computationally proficient scientists. We created OmicsVolcano, an interactive open-source software tool to enable visualization and exploration of high-throughput biological data, while highlighting features of interest using a volcano plot interface. In contrast to existing tools, our software and user-interface design allow it to be used without requiring any programming skills to generate high-quality and presentation-ready images.
MeSH term(s)	Computational Biology ; Gene Expression Profiling ; Genomics ; User-Computer Interface
Language	English
Publishing date	2021-01-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2020.100279
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Article ; Online: Mutational rescue of the activity of high-fidelity Cas9 enzymes.

Vos, Pascal D / Gandadireja, Andrianto P / Rossetti, Giulia / Siira, Stefan J / Mantegna, Jessica L / Filipovska, Aleksandra / Rackham, Oliver

Cell reports methods

2024 Volume 4, Issue 4, Page(s) 100756

Abstract: Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have ... ...

Abstract	Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.
MeSH term(s)	Humans ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; HEK293 Cells ; Mutation ; Endonucleases/genetics ; Endonucleases/metabolism
Chemical Substances	CRISPR-Associated Protein 9 (EC 3.1.-) ; Endonucleases (EC 3.1.-)
Language	English
Publishing date	2024-04-11
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2667-2375
ISSN (online)	2667-2375
DOI	10.1016/j.crmeth.2024.100756
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Article ; Online: Investigating Mitochondrial Transcriptomes and RNA Processing Using Circular RNA Sequencing.

Kuznetsova, Irina / Rackham, Oliver / Filipovska, Aleksandra

Methods in molecular biology (Clifton, N.J.)

2020 Volume 2192, Page(s) 43–57

Abstract: Transcriptomic technologies have revolutionized the study of gene expression and RNA biology. Different RNA sequencing methods enable the analyses of diverse species of transcripts, including their abundance, processing, stability, and other specific ... ...

Abstract	Transcriptomic technologies have revolutionized the study of gene expression and RNA biology. Different RNA sequencing methods enable the analyses of diverse species of transcripts, including their abundance, processing, stability, and other specific features. Mitochondrial transcriptomics has benefited from these technologies that have revealed the surprising complexity of its RNAs. Here we describe a method based upon cyclization of mitochondrial RNAs and next generation sequencing to analyze the steady-state levels and sizes of mitochondrial RNAs, their degradation products, as well as their processing intermediates by capturing both 5' and 3' ends of transcripts.
MeSH term(s)	Animals ; Computational Biology/methods ; Gene Expression Profiling/methods ; Genome, Mitochondrial ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Mice ; Mitochondria/genetics ; RNA Processing, Post-Transcriptional/genetics ; RNA, Circular/genetics ; RNA, Circular/metabolism ; RNA, Mitochondrial/genetics ; RNA, Mitochondrial/metabolism ; Rats ; Sequence Analysis, RNA/methods ; Software ; Transcriptome
Chemical Substances	RNA, Circular ; RNA, Mitochondrial
Language	English
Publishing date	2020-11-23
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0834-0_4
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Article ; Online: Preventative therapeutic approaches for hypertrophic cardiomyopathy.

Solomon, Tanya / Filipovska, Aleksandra / Hool, Livia / Viola, Helena

The Journal of physiology

2020 Volume 599, Issue 14, Page(s) 3495–3512

Abstract: Sarcomeric gene mutations are associated with the development of hypertrophic cardiomyopathy (HCM). Current drug therapeutics for HCM patients are effective in relieving symptoms, but do not prevent or reverse disease progression. Moreover, due to ... ...

Abstract	Sarcomeric gene mutations are associated with the development of hypertrophic cardiomyopathy (HCM). Current drug therapeutics for HCM patients are effective in relieving symptoms, but do not prevent or reverse disease progression. Moreover, due to heterogeneity in the clinical manifestations of the disease, patients experience variable outcomes in response to therapeutics. Mechanistically, alterations in calcium handling, sarcomeric disorganization, energy metabolism and contractility participate in HCM disease progression. While some similarities exist, each mutation appears to lead to mutation-specific pathophysiology. Furthermore, these alterations may precede or proceed development of the pathology. This review assesses the efficacy of HCM therapeutics from studies performed in animal models of HCM and human clinical trials. Evidence suggests that a preventative rather than corrective therapeutic approach may be more efficacious in the treatment of HCM. In addition, a clear understanding of mutation-specific mechanisms may assist in informing the most effective therapeutic mode of action.
MeSH term(s)	Animals ; Calcium/metabolism ; Cardiomyopathy, Hypertrophic/drug therapy ; Cardiomyopathy, Hypertrophic/genetics ; Energy Metabolism ; Humans ; Mutation ; Sarcomeres/metabolism
Chemical Substances	Calcium (SY7Q814VUP)
Language	English
Publishing date	2020-09-07
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	3115-x
ISSN	1469-7793 ; 0022-3751
ISSN (online)	1469-7793
ISSN	0022-3751
DOI	10.1113/JP279410
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Article ; Online: In silico evolution of nucleic acid-binding proteins from a nonfunctional scaffold.

Raven, Samuel A / Payne, Blake / Bruce, Mitchell / Filipovska, Aleksandra / Rackham, Oliver

Nature chemical biology

2022 Volume 18, Issue 4, Page(s) 403–411

Abstract: Directed evolution emulates the process of natural selection to produce proteins with improved or altered functions. These approaches have proven to be very powerful but are technically challenging and particularly time and resource intensive. To bypass ... ...

Abstract	Directed evolution emulates the process of natural selection to produce proteins with improved or altered functions. These approaches have proven to be very powerful but are technically challenging and particularly time and resource intensive. To bypass these limitations, we constructed a system to perform the entire process of directed evolution in silico. We employed iterative computational cycles of mutation and evaluation to predict mutations that confer high-affinity binding activities for DNA and RNA to an initial de novo designed protein with no inherent function. Beneficial mutations revealed modes of nucleic acid recognition not previously observed in natural proteins, highlighting the ability of computational directed evolution to access new molecular functions. Furthermore, the process by which new functions were obtained closely resembles natural evolution and can provide insights into the contributions of mutation rate, population size and selective pressure on functionalization of macromolecules in nature.
MeSH term(s)	DNA/chemistry ; Directed Molecular Evolution ; Mutation ; Nucleic Acids ; Proteins/chemistry ; RNA
Chemical Substances	Nucleic Acids ; Proteins ; RNA (63231-63-0) ; DNA (9007-49-2)
Language	English
Publishing date	2022-02-24
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2202962-X
ISSN	1552-4469 ; 1552-4450
ISSN (online)	1552-4469
ISSN	1552-4450
DOI	10.1038/s41589-022-00967-y
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Article ; Online: Mitochondrial gene expression is required for platelet function and blood clotting.

Richman, Tara R / Ermer, Judith A / Baker, Jessica / Siira, Stefan J / Kile, Benjamin T / Linden, Matthew D / Rackham, Oliver / Filipovska, Aleksandra

Cell reports

2023 Volume 42, Issue 11, Page(s) 113312

Abstract: Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative ... ...

Abstract	Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative phosphorylation system to produce energy required for thrombosis. The autonomy of the mitochondrial gene expression machinery from the nucleus is unclear, and platelets provide a valuable model to understand its importance in anucleate cells. Here, we conditionally delete Elac2, Ptcd1, or Mtif3 in platelets, which are essential for mitochondrial gene expression at the level of RNA processing, stability, or translation, respectively. Loss of ELAC2, PTCD1, or MTIF3 leads to increased megakaryocyte ploidy, elevated circulating levels of reticulated platelets, thrombocytopenia, and consequent extended bleeding time. Impaired mitochondrial gene expression reduces agonist-induced platelet activation. Transcriptomic and proteomic analyses show that mitochondrial gene expression is required for fibrinolysis, hemostasis, and blood coagulation in response to injury.
MeSH term(s)	Humans ; Genes, Mitochondrial ; Proteomics ; Hemostasis/physiology ; Blood Coagulation ; Blood Platelets/metabolism ; Megakaryocytes/metabolism ; Thrombosis ; Gene Expression ; Mitochondrial Proteins/metabolism
Chemical Substances	PTCD1 protein, human ; Mitochondrial Proteins
Language	English
Publishing date	2023-10-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2023.113312
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