Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

Abstract

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released.RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation.The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex

“…PRO , but not TbPRMT1 ENZ , is reportedly phosphorylated (8,45), bound to mRNA (46), and trafficked to stress granules in starved trypanosomes (47). This suggests that TbPRMT1 PRO could be, in certain situations, operating independently of TbPRMT1 ENZ , possibly as a homodimer (Fig.…”

Genome-wide RNAi selection identifies a regulator of transmission stage-enriched gene families and cell-type differentiation in Trypanosoma brucei

“…Co-staining of the cells with AMCA which stains the cell surface and flagellar pocket demonstrated that the REG9.1 signal was only partly overlapping with the flagellar pocket (Fig 9B) and ‘no first antibody’ controls confirmed the signal was not caused by secondary antibody recognition of host antibodies in the flagellar pocket. Since REG9.1 has been detected in procyclic form stress granules by proteomic analysis [34], we explored whether REG9.1 coalesced from its uniform cytoplasmic distribution in procyclic forms to discrete foci upon starvation or heat shock. Under starvation conditions (2h in PBS) REG9.1 co-localised partially with the stress granule marker Scd6 [35] (S5A Fig), but this was not evident with heat shock, where Scd6 signal was also not obvious.…”

Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms

“…Lysis of trypanosomes in the presence of 1% Triton X-100 results in trapping of structures with a diameter of more than 24nm within the microtubule corset [21]. (For comparison, a ribosome is just under 30nm across.)…”

“…Some mRNAs that were excluded (<20%) from heat shock granules were also similarly absent from starvation granules [21]. Presumably these encode products that are required to recover from both starvation and heat shock.…”