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ABSTRACT: Pre-mRNA processing complex in Trypanosoma brucei: structural assembly and function of core polyA factors

PROVIDER: PRJNA845072 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19536174_1.fastq.gz	Fastqsanger.gz
	SRR19536174_2.fastq.gz	Fastqsanger.gz
	SRR19536175_1.fastq.gz	Fastqsanger.gz
	SRR19536175_2.fastq.gz	Fastqsanger.gz
	SRR19536176_1.fastq.gz	Fastqsanger.gz

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Pre-mRNA processing complex in Trypanosoma brucei: structural assembly and function of core polyA factors

Project description:Pre mRNA 3 end processing is unique in trypanosomes as it involves polycistronic transcription, polyadenylation coupled to trans splicing and elusive polyadenylation signals. The mechanism is poorly understood. Here we reported that nine putative polyA factors from Trypanosoma brucei form a stable complex which then subsequently develops into two tight subcomplexes. One contains the proteins tbCPSF160, tbCPSF30, tbFip1 and tbWdr33 with tbCPSF160 as the scaffold; while the other contains tbCPSF100, tbCPSF73, tbSYMK and two trypanosome unique proteins, designated CPF30 and CPF15. No CstF homologs were found. The protein tbCPSF100 seems to coordinate the association of these two subcomplexes by interacting with tbCPSF160. RNAi knockdown of these polyA factors resulted in RNA processing disorders and variable disruption of the complex and RNA processing disorders. Depletion of either factors caused dicistronic transcripts formation; depletion of either tbCPSF30, tbFip1 or tbWdr33 caused increased usage of proximal polyadenylation sites of the transcripts; depletion of tbCPSF30 caused mRNA polyA tail shortening. This indicates that these two polysA factors have a critical role in polyadenylation site selection. Genome-wide UV crosslinking assays showed that tbCPSF30 and tbFip1 have strong RNA-binding activity in this complex. Altogether, these data indicate that the 3 end processing complex of T. brucei is more like the human CPSF homolog. However, it displays trypanosome specific features in composition, structural assembly and function of components, which facilitate a common, yet unique, cleavage polyadenylation reaction.

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Histone divergence in Trypanosoma brucei results in unique alterations in nucleosome structure

Project description:Eukaryotes have an array of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from Kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically characterised nucleosome core particles (NCPs) from the Kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened DNA binding overall. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.

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The Myb domain of TbRAP1 plays an important role in subtelomeric gene expression

Project description:We examine the function of the TbRAP1 Myb domain in gene expression regulation in Trypanosoma brucei that causes human African trypanosomiasis. TbRAP1 is required for normal VSG monoallelic expression, a key aspect of antigenic variation that is used by T. brucei to evade the host immune response.

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Project description:We examine the function of the TbRAP1 DB domain in gene expression regulation in Trypanosoma brucei that causes human African trypanosomiasis. TbRAP1 is required for normal VSG monoallelic expression, a key aspect of antigenic variation that is used by T. brucei to evade the host immune response.

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Project description:Raw file submission for publication 'Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei'

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Project description:Trypanosoma brucei Lister 427 bloodstream forms were cultured in HMI-11 medium. Total RNA was prepared using Qiagen RNAeasy kits for single sample RNAseq to estimate VSG mRNA abundance (and not to reconstruct the transcriptome). The cDNA libraries were prepared and sequenced at the Beijing Genomics Institute (Shenzhen, China). Polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers sheared and size selected for fragments ~200 bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2. RNAseq of the resulting libraries was used for the determination of transcript abundances. Sequencing was performed on an Illumina Hiseq 2000 (Illumina, CA) platform and 90 base paired end reads obtained. Four samples were analysed: 1. Trypanosoma brucei Lister 427 expressing VSG2 2. Trypanosoma brucei Lister 427 expressing VSG6 3. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 28 days after electroporation 4. Trypanosoma brucei Lister 427 expressing VSG6 and a VSG2 transgender located in the active bloodstream expression site 44 days after electroporation.

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