Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression

Abstract

The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component.

Nuclear Phosphatidylinositol 5-Phosphatase Is Essential for Allelic Exclusion of Variant Surface Glycoprotein Genes in Trypanosomes

“…However, alternative functions for these proteins in ES regulation cannot be excluded. Similarly, reverse IPs and localizations also validated PIP5Pase's interaction with NUP144, which is a component of the nuclear lamina and highlights the role of nuclear lamina and perhaps chromatin subnuclear compartmentalization in ES regulation (18,37,38). HELT1, a hypothetical protein with a partial helicase domain, and CRK9, which has kinase activity (39), were also validated and suggest other regulatory processes involved in ES transcription control.…”

“…Silent ESs are enriched in histones such as H1, H3, H3V, and base J (30,31,34), chromatin-modifying enzymes such as DOT1B methyltransferase (10), and BDFs (12), which suggests a compact chromatin structure involved in ES silencing. Subnuclear location may also affect ES silencing, as implied by loss of VSG silencing upon NUP-1 and NUP-2 knockdown (18,37) and immunoprecipitations by PIP5Pase or RAP1 of nuclear lamina and nuclear pore proteins (e.g., NUP-1, NUP158, and NUP144). Thus, PIP5Pase and RAP1 may function in concert with those proteins to establish a chromatin structure that represses RNAP I elongation through VSG genes in silent ESs.…”

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex

“…5 Additionally, they have retained similar functions to those in other eukarya, by creating heterochromatin free zones around nuclear pores that are evident by electron microscopy in all eukaryotic nuclei, as well as recapitulating interactions with the spindle organizer. 4,38,[79][80][81] Thus, in conjunction with divergent lamin-like proteins, 35,82 and unconventional kinetochores, 83 it seems that the trypanosome nucleus uses unique protein complexes in parallel with conserved core elements such as the spindle microtubules and outer kinetochore components, Ndc80/Nuf2 to facilitate trypanosome biology. 84,85 Did trypanosomes retain an ancient symmetric assembly or reconfigure their molecular biology through the loss of introns and individual gene promoters (Fig.…”

Specialising the parasite nucleus: Pores, lamins, chromatin, and diversity

“…In trypanosomes, two large coiled-coil proteins, NUP-1 (450 kDa) and NUP-2 (170 kDa), are major components of the nuclear lamina that are involved in maintaining silent VSG genes at subtelomeric expression sites in a state of very low transcriptional activity [21, 22]. Additionally, both also participate in repression of procyclin, the major antigen expressed in the insect stage, in the mammalian-infective form; significantly, both VSGs and procyclin are transcribed by RNA polymerase I, which sets both of these loci apart from the bulk of protein coding genes, which are transcribed by RNA Pol II.…”

“…It remains to be understood how the mechanisms of transcription, chromatin modification, and silencing connects with this lamina at the molecular level, but at the cellular level, the role in maintaining a structure that allows segregation of chromatin into peripheral heterochromatin is likely critical. Further, as NUP-1 and NUP-2 are conserved across trypanosomes, this suggests a similar system is present in many pathogenic protozoa [14, 22]. In every important structural and functional sense examined, NUP-1 and NUP-2 both behave similarly to lamins.…”