Glucocerebrosidase 1 () mutations responsible for Gaucher disease (GD) are the most common genetic risk factor for Parkinson's disease (PD). Although the genetic link between GD and PD is well established, the underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a sphingolipid accumulating in GD, mediates PD pathology in -associated PD. We show that, whereas GD-related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote α-synuclein aggregation, glucosylsphingosine triggers the formation of oligomeric α-synuclein species capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex [ mutant (N370S, L444P, KO) crossed to α-synuclein transgenics], we show that mutations predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide accumulations colocalized with α-synuclein pathology. These findings indicate that glucosylsphingosine promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers. Parkinson's disease (PD) is a prevalent neurodegenerative disorder in the aging population. Glucocerebrosidase 1 mutations, which cause Gaucher disease, are the most common genetic risk factor for PD, underscoring the importance of delineating the mechanisms underlying mutant -associated PD. We show that lipids accumulating in Gaucher disease, especially glucosylsphingosine, play a key role in PD pathology in the brain. These data indicate that ASAH1 (acid ceramidase 1) and GBA2 (glucocerebrosidase 2) enzymes that mediate glucosylsphingosine production and metabolism are attractive therapeutic targets for treating mutant-associated PD.

Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3 portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.One of the most intriguing aspects in protozoa of the family Trypanosomatidae, which include African and South American trypanosomes and Leishmania, remains the regulation of gene expression (5). It appears that these organisms do not use transcription initiation as a major regulatory step to control the output of mRNA on a per gene basis; the majority of proteincoding genes are organized as polycistronic rather than monocistronic transcription units. Indeed, the 5Ј ends of all mature mRNAs are formed by trans-splicing, an RNA processing reaction, rather than by transcription initiation as in most eukaryotic organisms.Another curiosity in trypanosomatids is the apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Although transcription is performed by three RNA polymerases with ␣-amanitin sensitivities similar to those of higher eukaryotic Pol I, II, and III, thus far it has been challenging to map Pol II transcription initiation sites for protein-coding genes (5). To date, there is a single report where transcriptional analysis of Leishmania major chromosome 1 suggests the presence of a bidirectional Pol II promoter (26). The only other characterized Pol II-dependent promoter drives expression of the spliced leader (SL) RNA (11). Genetic and biochemical studies delineated various promoter elements of the SL RNA gene and led to the isolation of the first transcription factor (promoter-binding protein 1 [PBP-1]) in trypanosomatids (8). PBP-1 interacts with the promoter element located between 60 and 80 bp upstream of the transcription start site. Interestingly, the 57-kDa subunit of PBP-1 is orthologous to the 50-kDa component of the small nuclear RNA-activating protein complex (SNAP c ), involved in transcription of human small nuclear RNA (snRNA) genes (8). Studi...

Many U-snRNAs contain a hypermodified 2,2,7-trimethylguanosine (TMG) cap structure, which is formed by post-transcriptional methylation of an m(7)G cap. At present, little is known about the maturation of U-snRNAs in trypanosomes. The current evidence is consistent with the primary transcript containing an m(7)G moiety, but it is not clear whether the conversion of m(7)G to TMG takes place in the cytoplasm or in the nucleus. To address this issue, we characterized the Trypanosoma brucei homologue of the trimethylguanosine synthase (TbTgs1), a 28kDa protein, which is mainly composed of the conserved catalytic domain and lacks a large N-terminal domain present in higher eukaryotes. A GFP fusion with TbTgs1 revealed that this protein localizes throughout the nucleoplasm, as well as in one or two dots outside the nucleolus and RNAi-mediated downregulation of TbTgs1 suggests that this protein is responsible for hypermethylation of the m(7)G cap of both snRNAs and snoRNAs.

SummaryThe Trypanosoma brucei genome is colonized by the site-specific non-LTR retrotransposon SLACS, or spliced leader-associated conserved sequence, which integrates exclusively into the spliced leader (SL) RNA genes. Although there is evidence that the RNA interference (RNAi) machinery regulates SLACS transcript levels, we do not know whether RNAi deficiency affects the genomic stability of SLACS, nor do we understand the mechanism of SLACS transcription. Here, we report that prolonged culturing of RNAi-deficient T. brucei cells, but not wild-type cells, results in genomic rearrangements of SLACS. Furthermore, two populations of SLACS transcripts persist in RNAi-deficient cells: a full-length transcript of approximately 7 kb and a heterogeneous population of small SLACS transcripts ranging in size from 450 to 550 nt. We provide evidence that SLACS transcription initiates at the +1 of the interrupted SL RNA gene and proceeds into the 5Ј UTR and open reading frame 1 (ORF1). This transcription is carried out by an RNA polymerase with a-amanitin sensitivity reminiscent of SL RNA synthesis and is dependent on the SL RNA promoter. Additionally, we show that both sense and antisense small SLACS transcripts originate from ORF1 and that they are associated with proteins in vivo. We speculate that the small SLACS transcripts serve as substrates for the production of siRNAs to regulate SLACS expression.

Capping of the pre-mRNA 5' end by addition a monomethylated guanosine cap (m(7)G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m(7)G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m(7)G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes.