SummaryTrypanosoma brucei is an extracellular parasite that causes sleeping sickness. In mammalian hosts, trypanosomes are thought to exist in two major niches: early in infection, they populate the blood; later, they breach the blood-brain barrier. Working with a well-established mouse model, we discovered that adipose tissue constitutes a third major reservoir for T. brucei. Parasites from adipose tissue, here termed adipose tissue forms (ATFs), can replicate and were capable of infecting a naive animal. ATFs were transcriptionally distinct from bloodstream forms, and the genes upregulated included putative fatty acid β-oxidation enzymes. Consistent with this, ATFs were able to utilize exogenous myristate and form β-oxidation intermediates, suggesting that ATF parasites can use fatty acids as an external carbon source. These findings identify the adipose tissue as a niche for T. brucei during its mammalian life cycle and could potentially explain the weight loss associated with sleeping sickness.

Highlights d RNase H2 efficiently processes R-loops and rNMPs postreplicatively d rNMPs processed by RNase H2 in S phase require Rad52mediated repair d RNase H1 functions independently of the cell cycle d RNase H1 responds to R-loop-induced stress

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

16Trypanosoma brucei causes African sleeping sickness, a fatal human disease. Its differentiation 17 from replicative slender form into quiescent stumpy form promotes host survival and parasite 18 transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation. To 19 determine whether lncRNAs are involved in parasite differentiation we used RNAseq to survey 20 the T. brucei lncRNA gene repertoire, identifying 1,428 previously uncharacterized lncRNA 21 genes. We analysed grumpy, a lncRNA located immediately upstream of an RNA-binding 22 protein that is a key differentiation regulator. Grumpy over-expression resulted in premature 23 parasite differentiation into the quiescent stumpy form, and subsequent impairment of in vivo 24 infection, decreasing parasite load in the mammalian host, and increasing host survival. Our 25 analyses suggest Grumpy is one of many lncRNA that modulate parasite-host interactions, and 26 lncRNA roles in cell differentiation are probably commonplace in T. brucei. 27 Keywords: 28Trypanosoma brucei, sleeping sickness, parasite, long non-coding RNAs, differentiation, 29 stumpy forms. 30 31 32