Proteomic analysis defines the interactome of telomerase in the protozoan parasite, Trypanosoma brucei

Abstract

Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.

Molecular and Evolutionary Analysis of RNA–Protein Interactions in Telomerase Regulation

“…The telomerase-interacting proteins have not been well studied in relation to parasitic protozoa. A recent interactome study of T. brucei TERT ( Tb TERT) carried out by our group identified conserved and novel protein interactions in the T. brucei telomerase complex [ 32 ]. Conserved interactions included an association with a variety of molecular chaperones, including the mitochondrial chaperone HSP60 [ 47 , 48 ].…”

“…Conserved interactions included an association with a variety of molecular chaperones, including the mitochondrial chaperone HSP60 [ 47 , 48 ]. Another notably conserved interaction was with a T. brucei la protein (Tb927.10.2370), which is a potential homolog of p65 from Tetrahymena [ 32 ]. The TR in humans contains an H/ACA snoRNA domain that is replaced by a C/D box domain in T. brucei [ 33 ] TR.…”

“…Subsequent AP-MS profiling experiments revealed a more extensive list of interacting proteins, including core H/ACA-box-binding proteins and other unique RNA-binding proteins discussed earlier [ 20 ]. AP-MS techniques have also been successfully utilized to study the global interactors of telomerase across diverse eukaryotes, including T. thermophila , yeast, and T. brucei [ 25 , 26 , 27 , 31 , 32 ].…”

“…A unique feature of kinetoplastid TR molecules is the presence of a C/D snoRNA domain [ 33 , 63 ]. Core C/D-box-binding proteins have been found to be associated with the telomerase complex in T. brucei [ 32 , 33 ]. The exact role of these proteins in TR biogenesis and processing is unknown, but they replace the H/ACA box domain found in humans.…”

“…This high-resolution structure demonstrated that NHP2, NOP10, TCAB1, GAR1, and dyskerin are important components of the telomerase holoenzyme when bound to its substrate. T. brucei Q38AL4 [32,33] The cryo-EM structure of the yeast telomerase holoenzyme remains to be solved. However, key interacting proteins of telomerase in Saccharomyces cerevisiae (S. cerevisiae) have been identified using biochemical and mass-spectrometry-based approaches.…”