Investigation of Trans-factors responsible for the developmentally linked instability of a highly regulated mRNA in Trypanosoma brucei

*Trypanosoma brucei*, a protozoan pathogen of both humans and livestock, has a number of divergent features arising from its history as an early branching eukaryote. One such feature is an RNA polymerase II gene expression system in which genes of diverse functions and expression profiles are transcribed polycistronically at roughly equal rates. With the reduction of gene specific regulation at the point of transcription initiation, trypanosomes provide a model for post-transcriptional gene regulation. One important mechanism is the regulated instability of mRNAs, the coordination of which is only partly understood. To identify responsible factors, a reporter based on the regulatory 3’ untranslated region of the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) mRNA, highly unstable in the procyclic developmental stage, was used in an RNAi library screen. A number of factors potentially responsible were identified, of which six were positively validated as being necessary for developmental regulation of the reporter mRNA, and three were successful at significant stabilisation of native GPI-PLC mRNA. Observing the sizes of stabilised reporter and GPI-PLC transcripts, together with the identity of annotated candidates indicated that stabilisation was achieved by the disruption of correct splicing causing the loss of regulatory elements. That none of the candidate genes achieved significant stabilisation without altering the sequence of the regulatory UTR is suggestive of a complex profile of interacting factors, none of which is necessary on its own for normal regulation, that important factors are essential or that factors are still capable of mediating regulation after depletion by RNAi. In addition expression of RNA-guided DNA endonuclease Cas9 was performed in *T. brucei* and the CRISPR system was assessed for future forward genetic screens, the results of which were highly encouraging. The first method assessed involved single target knockout by targeted Cas-9 action which proved sufficiently effective to suggest feasible CRISPR screens utilising a guide RNA library. Secondly, a dramatically increased rate of integration was observed to Cas9 targeted locations, demonstrated through multiple transfections at increased efficiency in terms of proportion of cells transfected, including single-step double knockouts in bloodstream form cells at rate of 0.033%, an efficiency in excess of even single transfections without Cas9. This would allow the transfection of RNAi libraries at a rate far above conventional approaches, ensuring maximal coverage in future screens. In these tests, double knockouts and derived genomic material were also generated for use in a collaborative project looking at the distribution in DNA of the unusual modified base-J in the absence of its deposition machinery, at a rate greatly accelerated by the Cas9 approach.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Barrass, Stephen

Advisor dc:contributor.advisor

• Carrington, Mark

• CRISPR
• Developmental regulation
• Forward genetics
• Kinetoplastid
• mRNA regulation
• Parasite lifecycle
• Post-transcriptional regulation
• RNAi library screening
• Trypanosoma
• Trypanosoma brucei

Licence

• https://www.repository.cam.ac.uk/bitstreams/a09e3376-8894-44f1-9d38-d49dbaca9996/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113202

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375631