Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

Klebanov-Akopyan, Olga; Mishra, Amartya; Glousker, Galina; Tzfati, Yehuda; Shlomai, Joseph

doi:10.1093/nar/gky597

Cited by 14 publications

(20 citation statements)

References 78 publications

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“…Expression of recombinant GST‐tagged DRBD18 was carried out as described earlier (Lott et al., 2015). For His‐Mtr2 (Tb927.7.5760), the ORF was cloned into the MCS‐1 of pETDuet‐1vector (with MSC2 left empty), with frameshift correction by insertion of single nucleotide before the start codon (Kafková et al., 2017) and was purified using immobilized metal affinity chromatography (IMAC) as described earlier (Klebanov‐Akopyan et al., 2018). For GST pulldown assays, all recombinant plasmids including pGEX4T‐1, were transformed into E .…”

Section: Methodsmentioning

confidence: 99%

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Mishra

Kaur

McSkimming

et al. 2021

Molecular Microbiology

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Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron‐containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole‐cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

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Section: Methodsmentioning

confidence: 99%

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Mishra

Kaur

McSkimming

et al. 2021

Molecular Microbiology

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“…Expression of recombinant GST-tagged DRBD18 was carried out as described earlier (Lott et al, 2015). For His-Mtr2 (Tb927.7.5760), the ORF was cloned into the MCS-1 of pETDuet-1vector (with MSC2 left empty), with frame shift correction by insertion of single nucleotide before the start codon (Kafková et al, 2017) and was purified using immobilized metal affinity chromatography (IMAC) as described earlier (Klebanov-Akopyan et al, 2018). For GST pulldown assays, all recombinant plasmids including pGEX4T-1, were transformed into Escherichia coli BL21 strain, and protein expression was induced by addition of 0.5 mM isopropyl 1-thio-Dgalactopyranoside (IPTG) and subsequent growth at 22°C overnight.…”

Section: Protein Expression and Gst Pulldown Assaymentioning

confidence: 99%

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Mishra

Kaur

McSkimming³

et al. 2021

Preprint

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Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

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“…Recently, it was shown that T. brucei UMSBP2, which is involved in kDNA replication and segregation, is also localized at telomeres. The RNAi system showed that this protein not only participates in nuclear division but also plays a role in the coordinated replication of DNA-containing organelles 65 .…”

Section: Discussionmentioning

confidence: 99%

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship

Gonçalves¹,

Catta-Preta²,

Repolês³

et al. 2021

Sci Rep

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Angomonas deanei coevolves in a mutualistic relationship with a symbiotic bacterium that divides in synchronicity with other host cell structures. Trypanosomatid mitochondrial DNA is contained in the kinetoplast and is composed of thousands of interlocked DNA circles (kDNA). The arrangement of kDNA is related to the presence of histone-like proteins, known as KAPs (kinetoplast-associated proteins), that neutralize the negatively charged kDNA, thereby affecting the activity of mitochondrial enzymes involved in replication, transcription and repair. In this study, CRISPR-Cas9 was used to delete both alleles of the A. deanei KAP4 gene. Gene-deficient mutants exhibited high compaction of the kDNA network and displayed atypical phenotypes, such as the appearance of a filamentous symbionts, cells containing two nuclei and one kinetoplast, and division blocks. Treatment with cisplatin and UV showed that Δkap4 null mutants were not more sensitive to DNA damage and repair than wild-type cells. Notably, lesions caused by these genotoxic agents in the mitochondrial DNA could be repaired, suggesting that the kDNA in the kinetoplast of trypanosomatids has unique repair mechanisms. Taken together, our data indicate that although KAP4 is not an essential protein, it plays important roles in kDNA arrangement and replication, as well as in the maintenance of symbiosis.

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Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

Cited by 14 publications

References 78 publications

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

Importance of Angomonas deanei KAP4 for kDNA arrangement, cell division and maintenance of the host-bacterium relationship

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