Developmentally Regulated Novel Non-coding Anti-sense Regulators of mRNA Translation in Trypanosoma brucei

Figures

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  Figure 1. TBsRNAs Are Developmentally Regulated (A) Enrichment of ncRNAs from T. brucei cell extracts. Whole cell extracts from 2 × 109 cells were prepared and depleted of ribosomes, as described in (Chikne et al., 2016; Rajan et al., 2019a, 2019b). RNA was extracted from PRS and total cell lysate (T), separated on a 10% denaturing gel, and stained with ethidium bromide. (B) Example of the RNA composition of the PRS library. Pie diagram depicts the relative abundance of various ncRNA types in two libraries. Approximately, 13 different PRS libraries were used to validate the existence of the 62 ncRNA described in this study. (C) Representative snapshot coverage of two novel TBsRNAs on their corresponding chromosome locus, given in RPKM. (D) Distribution of the ncRNA reads across the pre-rRNA given in Reads per kilobase per million (RPKM). The arrows indicate the direction of transcription. The domains of the pre-rRNA are indicated. (E) Northern analysis of ncRNAs. Total T. brucei RNA (20μg) was separated on denaturing gels and probed with 32P-labeled anti-sense RNA probe. 32P-labeled pBR322 DNA MspI digest was used as a size marker. (F) TBsRNAs are developmentally regulated. The volcano…

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  Figure 2. Localization of TBsRNAs (A) Fractionation of TBsRNA. Whole cell extract from procyclic cells (2 × 109) was fractionated on (15–50%) sucrose gradient at 70,000 rpm for 1.5 hr using a Beckman SW41 rotor. 400 μL fractions were collected using the ISCO gradient fractionation system. The fractions were deproteinized and the RNA was separated on a 6% or 10% polyacrylamide-denaturing gel and subjected to Northern analysis using anti-sense RNA probes hybridizing to TBsRNAs, as indicated. The optical density of the fractions is presented. (B) High-resolution fluorescence in situ hybridization coupled with immunofluorescence was performed for the indicated RNAs (green) and NHP2 (red). DNA was stained with DAPI (blue). The scale bar is indicated.

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  Figure 3. T.brucei ncRNA Interactome (A) Schematic presentation of the ncRNA interactome identification protocol. The scheme illustrates the in vivo AMT-psoralen UV cross-linking treatment, extract preparation, fractionation, RNA extraction, mild fragmentation, ligation, and the RNA library preparation methodology. (B) Enrichment of intermolecular RNA-RNA hybrids in ncRNA interactome upon ligation. The results show that the percentage of chimeric RNA increase from negligible to nearly 4% following ligation (left) and indicate the percentage of intermolecular cross-links in two replicates (right). (C) Ciros plot representing the known interactions of U snRNA in T. brucei following ligation of cross-linked RNAs. (D) Chimeric snoRNA-pre rRNA. The potential for base-pairing of TB10Cs1C4 with the pre-rRNA, and the coverage of ligation products between these RNAs in RPKM across the pre-rRNA are presented. An example of a chimeric RNA sequence and its target snoRNA is shown, demonstrating that ligation between the RNAs takes place close to the interaction domain. The different domains of the pre-rRNA are indicated.

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  Figure 4. T.brucei TBsRNA Interactome (A) (i) Enrichment of RNA-RNA hybrids in the ncRNA interactome upon ligation in various replicates. (ii) The total number of TBsRNA-mRNA chimeric reads obtained in each library. (B) Gene ontology of mRNAs cross-linked with TBsRNA. (C) Circos plots representing the interactions of TBsRNA with mRNAs in T. brucei upon ligation of cross-linked RNAs.

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  Figure 5. TBsRNA-33 Is an Anti-Sense Regulator of T. bruceiPif1 mRNA (A) TBsRNA-33 interacts with Pif1 mRNA. (i) Schematic representation of the potential base-pairing between TBsRNA-33 and Pif1 mRNA. The arrows indicated the positions of primers used in (iii) to amplify the chimeric RNA. (ii) The sequence of chimeric RNA formed by the interaction between TBsRNA-33 and Pif1 mRNA 3′ UTR region is indicated from the PRS interactome library. (iii) Identification of TBsRNA-33 ligated to Pif1 mRNA 3′ UTR by RT-PCR. Total RNA was prepared using the protocol described in (Figure 3A), and the cDNA was subjected to gene specific PCR using a forward primer from TBsRNA-33, and reverse primers corresponding to different regions of Pif1 mRNA as indicated by the arrows in (i). (B) TBsRNA-33 silencing. Cells carrying the silencing construct for TBsRNA-33 were silenced for the indicated days, and the RNA was subjected to Northern analysis with the indicated anti-sense probes. (C) Pif1 mRNA is not increased following TBsRNA-33 silencing. Total RNA (20 μg) was prepared from cells before and after TBsRNA-33 silencing, separated on a 1.2% agarose/formaldehyde gel and subjected to Northern analysis using RNA…

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  Figure 6. TBsRNA-33 Is an Anti-Sense Regulator of T. brucei Pif1 mRNA (A) Growth of cells upon TBsRNA-33 silencing. Uninduced cells carrying the silencing construct (-TET) were compared with cells induced for silencing (+TET) at 27°C. Data are presented as mean ± S.E.M. Experiments were done in triplicate (n = 3). (B) Growth of cells upon TBsRNA-33 silencing and pentamidine treatment. Uninduced cells carrying the silencing construct (-TET) were compared with cells induced for silencing (+TET) upon treatment with 20μM pentamidine. Data are presented as mean ± S.E.M. Experiments were done in triplicate (n = 3). (C). Silencing of TBsRNA-33 induces loss of mitochondrial membrane potential upon pentamidine treatment. Uninduced cells (-TET) and cells induced for silencing (+TET) upon treatment with 20μM pentamidine were harvested and treated with 150 nM TMRM in PBS with 0.2% glucose. The samples were incubated in the dark for 15 min at 27°C and then analyzed by FACS in PE channel. The cell count of cells with reduced ΔΨm is plotted comparing the cells before and after two days of silencing. Data are presented as mean ± S.E.M (left). Experiments were done in triplicate (n = 3). Student's t-test…

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  Figure 7. TBsRNA-33 Is an Anti-Sense Regulator of Tb927.11.13610, Rhodanese-like Domain-Containing Protein mRNA (A) TBsRNA-33 interacts with Tb927.11.13610 mRNA. Schematic representation of the potential base-pairing between TBsRNA-33 and Tb927.11.13610 mRNA. (B) The sequence of the chimeric RNA formed by the interaction between TBsRNA-33 and the Tb927.11.13610 CDS region is indicated based on the PRS interactome library. (C) Tb927.11.13610 mRNA is not increased upon TBsRNA-33 silencing. Total RNA (20 μg) was prepared from cells before and after TBsRNA-33 silencing, separated on a 1.2% agarose/formaldehyde gel and subjected to Northern analysis using RNA probes, as indicated. 7SL RNA served as a loading control. (D) Tb927.11.13610 translation is increased upon TBsRNA-33 silencing. Tb927.11.13610 was tagged with N-terminal MYC tag in cells carrying the silencing construct for TBsRNA-33 and were silenced for the indicated number of days. Whole cell lysates from induced (+TET) and uninduced (-TET) cultures were subjected to western analysis with the indicated antibodies (left). (E) The expression level for Tb927.11.13610-MYC is presented as the mean ± S.E.M. Experiments were performed in…

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  Figure 8. TBsRNA-37 Is an Anti-Sense Regulator of TyIF mRNA (A) TBsRNA-37 is encoded in the ITS7 of pre-rRNA. Schematic representation of the pre-rRNA locus from which TBsRNA-37 is derived is presented. (B) TBsRNA-37 interacts with TyIF mRNA. Schematic representation of the potential base-pairing between TBsRNA-37 and TyIF mRNA. (C) The sequence of chimeric RNA formed by the interaction between TBsRNA-37 and TyIF mRNA CDS region is indicated based on the PRS interactome library. (D) TBsRNA-37 silencing. Cells carrying the silencing construct for TBsRNA-37 were silenced for the indicated days, and the RNA was subjected to Northern analysis with the indicated anti-sense probes. (E) Growth of cells upon TBsRNA-37 silencing. Uninduced cells carrying the silencing construct (-TET) were compared with cells induced for silencing (+TET) at 27°C. Data are presented as mean ± S.E.M. Experiments were done in triplicate (n = 3). (F) TyIF mRNA is not decreased upon TBsRNA-37 silencing. Total RNA (20 μg) was prepared from cells before and after TBsRNA-37 silencing, separated on a 1.2% agarose/formaldehyde gel, and subjected to Northern analysis using RNA probes, as indicated. 7SL RNA served as loading…

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  Figure 9. Proposed Model for the Mechanism of TBsRNA-37 as an Anti-Sense Regulator in T. Brucei (A) TBsRNA-37 can potentially base-pair with mature rRNA. The coverage of ligation products of TBsRNA-37 across the pre-rRNA is shown from the PRS interactome library. The potential base-pairing of TBsRNA-37 with the srRNA-1 (sr1) is shown. The different domains of the pre-rRNA are indicated. (B) The proposed mechanism for TBsRNA-37 interaction with its target mRNA. The ncRNA TBsRNA-37 is processed from the pre-rRNA in the nucleolus, potentially base-pairs with sr1 during the biogenesis of pre-LSU and moves together with the LSU to be assembled into a mature ribosome in the cytoplasm. In the cytoplasm, TBsRNA-37 remains base-paired with sr1 and interacts by base pairing to the target mRNA to inhibit or enhance translation.