Olga V Makarova

Direct contact details

makarova

Tel: 0116 229 7103

Email: om13@le.ac.uk

Personal details

PhD, PhD

Career

  • PhD: Department of Biochemistry, University of Leicester, 1997-1999.
  • PhD: Institut fur Molekularbiologie und Tumorforschung, Marburg, Germany, 1997-1999.
  • Postdoctoral: Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany.
  • Lecturer, Department of Biochemistry 2004.

Research

  • Spliceosome Assembly
  • MS Analysis of Multi-Protein Complexes involved in Splicing
  • RNA Splicing in Malaria Plasmodium Falciparum

Pre-mRNA, a spliceosome, its parts and their interactions

Protein-coding genes in humans have unequalled coding potential among all eukaryotes. Most of them (>95%) are alternatively spliced, producing several protein isoforms.  These isoforms are specific for certain cell types or cell cycle stages, and their expression is regulated during development.

It is no longer a surprise that improper splicing of protein-coding gene transcripts is the underlying cause of 20% of genetic diseases. Cancer cells produce protein isoforms favourable for cell survival and invasion. Manipulating splicing is emerging as a new direction for therapies.

Five small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 and U6 constitute the primary parts of splicing machinery that are assisted by a whole army of additional auxiliary factors. The boundaries between exons and introns (splice sites, SS) are demarcated by the binding of U1 snRNP at the 5’SS and U2 snRNP at the 3’SS. The 5’SS and 3’SS come into close proximity by not yet well understood mechanism and form a complex known as pre-spliceosome. The tri-snRNP particle consisting of U4, U5, and U6 delivers parts of the spliceosomal catalytic core – U6 RNA and U5snRNP. The spliceosome rearranges itself through the consecutive action of DExD/H helicases to build a catalytic core for two transesterification reactions that will join exons and release intron. In total, the reaction requires the assembly of 5 small RNA molecules and about 200 proteins. These proteins present numerous targets for therapeutic interventions.

Our core expertise lies in the use of immunopurification and gradient fractionation to isolate functional complexes of interest and analyse them by mass spectrometry (MS).

Current projects

  • Spliceosome assembly

The nuclear role of the SMN protein.

The survival of motor neurons (SMN) protein is indispensable for normal organism development and its insufficiency is the underlying cause of Spinal Muscular Atrophy (SMA) disease. This protein is ubiquitously expressed in all cells, and the reason why the motor neurons are primarily affected in the disease is widely debated. The SMN protein is distributed throughout the cell cytoplasm and also localises to the nuclear structures known as gems. The function of SMN in the cytoplasm is well understood, with major roles including the assembly of uridine-rich small nuclear RNP particles (U snRNPs, major components of spliceosomes) biogenesis and the transport of mRNA in axons. In contrast, very little is known about the nuclear functions of SMN. We have recently identified SMN as a component of early spliceosomal complexes. Now, we propose to address its function in spliceosome assembly and study its interactions with the spliceosomal components.

PRPF40A

PRPF40A is a multidomain protein, which has been proposed  to bridge 5'SS with 3'SS. We are investigating the interactions of individual WW and FF domains to understand the mechanisms of intron bridging.

 

  • RNA splicing in malaria parasite Plasmodium falciparum

The mechanism by which PfCLK inhibitors disrupt splicing in the malaria parasite Plasmodium falciparum.

This project is collaboration with Andrew Tobin (MRC Toxicology Unit/Department of Cell Physiology and Pharmacology, Leicester). The Tobin lab has identified 36 protein kinases in the malaria parasite that are essential for parasite survival during the blood stage. Among these are the members of the CLK family of kinases that are proposed to play a vital role in RNA processing. In a programme co-funded by a Developmental Gap Fund from the MRC and an Open Foundation grant from GSK, the Tobin group is currently screening for inhibitors to the parasite CLK family (PfCLK1-4) with the aim of generating lead compounds.

One of the major barriers for exploiting the PfCLK family as a target in malaria is a serious lack of understanding of the role of this kinase family in parasite RNA splicing. This project is designed to address this issue by combining the expertise of our labs to apply molecular parasitology together with biochemical, chemical genetics, pharmacological tools, proteomic systems approaches and RNA sequencing to address the following key areas:

  • To establish the impact of the PfCLK family on splicing complex formation in the malaria parasite
  • To define the substrates and modes of action of the PfCLK family in the parasite splicing complex
  • To determine the biological programmes that are disrupted following inhibition of members of the PfCLK

In this way we will provide vital basic information on the biological role of the PfCLK family in RNA processing. In addition we will define the modes of action of PfCLKs inhibitors that will facilitate their development as therapeutic for malaria.

Characterisation of differences in splicing apparatus of humans and malaria parasite Plasmodium falciparum in order to identify molecular targets for drug screening.

The therapies based on splicing intervention have recently emerged as efficient ways of correcting gene expression. While the splicing mechanisms in humans are being actively characterized, the malaria splicing has not been studied in many details. This project aims to investigate the nature and characteristics of malaria parasite splicing. This will not only provide a basic understanding of splicing process in malaria but importantly will provide information for a therapeutic intervention strategy that targets parasite specific processes involved in splicing.  Currently, no laboratory is targeting RNA splicing in malaria drug discovery yet we know that it is an essential process and that key regulatory parasite specific mechanisms may provide an opportunity to develop parasite-specific inhibitors.

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Contact Details

Department of Molecular and Cell Biology
Henry Wellcome Building
Lancaster Road
Leicester
LE1 7RH (Postal)

LE1 7HB (Sat Nav/Online maps)

T:  +44(0)116 229 7038
F:  +44(0)116 229 7123
MolCellBiol@le.ac.uk

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Redfearn Lecture 2017

To Be Confirmed