Dissecting Trypanosoma brucei RRP44 function in the maturation of segmented ribosomal RNA using a regulated genetic complementation system

Figures

• 

  Figure 1.. Diagram of T. brucei pre-rRNA and major intermediates of its pre-rRNA processing pathway. (A) Summary of the previously known T. brucei pre-rRNA processing steps. After an initial cleavage at site A’, the 9.6 kb pre-rRNA is processed at site B1 in ITS1, separating the 3.4 kb small subunit (SSU) from the 5.8 kb large subunit (LSU) precursors. Subsequent processing of the SSU precursors involves cleavages at sites A0 and A1 for excision of the 5′-ETS, followed by removal of the 3′ ITS1 segment and cleavage at site A2 from the 2.6 kb fragment, generating the mature 18S SSU rRNA. The 5.8 kb LSU pre-rRNA is initially cleaved at site C in the ITS2, generating the 0.61 kb and 5.1 kb precursors, followed by a cleavage of the 5.1 kb pre-rRNA in ITS5 generating the 3.9 kb precursor. The 3′ segment of the 0.61 kb pre-rRNA is processed by 3′-to-5′ RNases to generate the mature 5.8S rRNA. The 3.9 kb is processed in ITS3 and ITS4 to generate the mature LSUα, SR1 and LSUβ. It is unknown if the SR2, SR6 and SR4 pre-rRNAs are processed directly from the 5.1 kb pre-RNA or if a pre-rRNA containing the SR2, SR6 and SR4 pre-rRNAs is generated by the cleavage of 5.1 kb pre-rRNA at site F1 the…

• 

  Figure 2.. Complementation of the endogenous TbRRP44 knockdown by an RNAi-resistant copy of the wild type TbRRP44 gene (TbRRP44RWT). (A) Growth curves of three clones expressing TbRRP44R-WT compared to the TbRRP44 conditional strain. The curves represent the average number of T. brucei cells per ml of cultures performed in triplicate with the respective standard deviation, during six days in presence (Induced/Tet+) or absence (Uninduced) of tetracycline, with daily cell count and culture dilution back to 106 cells/ml every second day. (B) Cell number ratio of the tetracycline-induced vs. uninduced cultures shown in (A). Colored bars represent the average number of cells of the triplicates and the respective standard deviation. (C) TbRRP44 mRNA levels from cultures induced with tetracycline for 2 days relative to cultures not treated with tetracycline as determined by quantitative RT-PCR normalized against TbGAPDH. The reactions were performed in triplicates and repeated at least twice. The y axis shows the normalized average of TbRRP44 mRNA levels with the respective standard deviation. (D) Analysis of knockdown of the endogenous TbRRP44 protein and expression of TbRRP44R-WT. Western blots…

• 

  Figure 3.. Evaluation of the complementation of TbRRP44 knockdown by RNAi-resistant TbRRP44 mutants. (A) Growth rate of strains transfected with the rescue plasmids pTbFIX-HA-RRP44R-WT, pTbFIX-HA-RRP44R-D140N, pTbFIX-HA-RRP44R-D511N, pTbFIX-HA-RRP44R-D140N/D511N, pTbFIX-HA-RRP44R-ΔNPIN. The curves represent the average number of T. brucei cells per ml of cultures performed in triplicate with the respective standard deviation, during four days in presence (Induced/Tet+) or absence (uninduced) of tetracycline, with dilution back to 106 cells/ml every second day. (B) Cell number ratio of the tetracycline-induced vs. uninduced cultures shown in (A). Colored bars represent the average number of cells of the triplicates and the respective standard deviation. The TbRRP44R-D140N mutant does not show growth phenotype while the TbRRP44R-D511N, TbRRP44R-D140N/D511N and TbRRP44R-ΔNPIN show growth phenotypes similar to the parental conditional strain. (C) Quantitative RT-PCR determination of the TbRRP44 mRNA levels from cultures induced with tetracycline for 2 days relative to cultures not treated with tetracycline normalized against TbGAPDH. The reactions were performed in triplicates and repeated at…

• 

  Figure 4.. Analysis of SSU pre-rRNA processing by Northern blots using probes complementary to the 5′-ETS and ITS1. The TbRRP44 variants are identified at the top and probes at the bottom of each panel, respectively. Cultures untreated and treated with tetracycline (Tet) are indicated by – and +, respectively. Diagrams of the parts that compose each pre-rRNA fragment detected by each probe are indicated at the left and right sides of the images. The precursors detected at lower levels are indicated in fainter colors. Green and red vertical lines on the diagrams indicate the position of the probes on each pre-rRNA intermediate. The central panels show an overlay of the two blots. Green and red bands indicate the precursors detected only by the probe on the left or on the right, respectively. Orange/yellow indicates the precursors detected by both probes. (A) Northern blots performed with probes 5ETS-a (left) and ITS1-a (right). Both probes detect the 3.5 kb SSU pre-rRNA. In addition, probe 5ETS-a detects a 0.8 kb fragment of the excised 5′-ETS and, probe ITS1-a detects a 2.5 kb band in parental TbRRP44 KD cells and in cells expressing non-complementing TbRRP44 mutants. (B) Northern blots…

• 

  Figure 5.. Primer extension analysis of ITS1 processing sites. Lanes – and + indicate the primer extension products of reverse transcriptase reactions performed with probe 5.8S-IRDye800 and RNA extracted from T. brucei cells uninduced (Tet–) and from cells induced with tetracycline (Tet+) for 48 h. A, C, G, T, indicate a dideoxynucleotide sequence ladder generated by DNA sequencing using the same probe and a DNA template containing the ITS1, 5.8S rRNA and ITS2 segment, which was run in parallel on 8% polyacrylamide-7 M urea gels. (A) Analysis of primer extension products after a 2 h and 40 min electrophoresis time. Two products of lower molecular weight differ by a single nucleotide and migrate at a position that should correspond to the 5′ end of the mature 5.S rRNA. In control cells, the product terminating at cytosine –1 is more intense while a weaker band is observed at adenine + 1 relative to the 5.8S rRNA 5′ end of the sequence used in this work derived from plasmid pGEM-ITS1/2-C14. Both bands show similar intensities in TbRRP44 knockdown cells. (B) Analysis of primer extension products as described in (A) except that the electrophoresis was run for 4 h and 30 min to better resolve…

• 

  Figure 6.. Analysis of LSU pre-rRNAs by Northern blots with probes hybridizing in the 5.8S rRNA and in ITS2. Northern blots with probes ITS1-b and ITS3 are also shown for comparison. Diagrams of the parts that compose the precursors detected by each probe are indicated at the left and right sides of the images. The pre-rRNAs detected at lower levels are indicated in fainter colors. The diagrams of the precursors 5.4ab kb and 4.3ab kb for which the 5′ and 3′ boundaries could not be assigned are marked in grey. Green and red vertical lines on the diagrams indicate the relative position of the probes on each pre-rRNA intermediate. In the Northern blot images, probes are identified at the bottom and the TbRRP44 variants at the top of each panel, respectively. Cultures untreated and treated with tetracycline (Tet) are indicated by – and +, respectively. The central panel shows an overlay of the two blots. Green and red bands indicate the precursors detected only by probe on the left or on the right, respectively. Orange/yellow indicates the precursors detected by both probes. (A) Northern blots performed with probes ITS1-b (left) and 5.8S (right). (B) Northern blots performed with probes ITS2-a…

• 

  Figure 7.. Analysis of LSU precursors by Northern blots with probes hybridizing in ITS3, LSUβ, ITS5 and ITS7. Northern blots with probes ITS2-a and 5ETS-b are also shown for comparison. Diagrams of the parts that compose each pre-rRNA fragment detected by each probe are indicated at the left and right sides of the images. The precursors detected at lower levels are indicated in fainter colors. The diagrams of the precursor 4.3ab kb for which the 5′ and 3′ boundaries could not be assigned are marked in grey. Green and red vertical lines on the diagrams indicate the relative position of the probes on each pre-rRNA intermediate. In the Northern blot images, probes are identified at the bottom and the strains at the top of each panel, respectively. Tet - and + indicate, respectively, the cultures uninduced and induced with tetracycline. The central panels show overlays of the two respective Northern blots shown at left and right. Green bands indicate the precursors that are detected only by the probes of the left panels, red on the right panels, and orange/yellow indicates the precursors detected by both probes. (A) Northern blots performed with probes ITS2-a (left) and ITS7 (right). (B)…

• 

  Figure 8.. Analysis of small pre-rRNA fragments by northern blot. (A) Diagram of the T. brucei full-length pre-rRNA and tentative pre-rRNAs processing steps of the LSU-B0 and LSU-B1 pre-rRNAs leading to formation of the 5.8S rRNA and small LSU rRNAs from the 3′ region. Positions of the cleavage sites are indicated above the pre-rRNA diagram. Green and red boxes indicate the relative position of the probes on each pre-rRNA intermediate. The 5.8 kb LSU-B0 precursor and the unusual 2.7 kb precursor cleaved at sites B0 and D1 can give rise to the 0.7 kb pre-rRNA following a cleavage at site C2. A 0.2 kb pre-rRNA could be formed either by 5′-3′ and 3′-5′ processing of the 0.7 kb pre-rRNA or by direct cleavages of the 5.8 kb, 2.7 kb and 0.7 kb pre-rRNAs at sites B1 and C1. Processing of the 5.7 kb LSU-B1 would follow a similar pathway. However, the 0.6 kb and 2.6 kb pre-rRNA (processing steps indicated by question marks) were not unequivocally detected in this work. Precursors identified in red were only detected in TbRRP44 deficient cells. (B) Northern blots performed with probes ITS1-b (left) and ITS2-b (right). (C) Northern blots performed with probes ITS1-b (left) and ITS2-d (right). (D)…

• 

  Figure 9.. Effect of TbRRP44 knockdown on snoRNAs and spliced leader synthesis. The strains are identified at the top of each panel. – and + indicate the samples from cultures uninduced and induced with tetracycline (Tet). Each blot was co-hybridized with a signal recognition particle RNA probe (7SL). The Northern blot with the probes to detect the U3 snoRNA and the spliced leader were performed with 3 μg of total RNA, while the blots for the snoRNAs Tb9Cs3C3 and Tb10Cs4C3 were performed with 6 μg of total RNA. (A) Analysis of snoRNA synthesis. The probes used in the Northern blots of the respective snoRNAs are identified on the left side. In control cells, two bands were detected for the U3 snoRNA. In TbRRP44 deficient cells, there is accumulation of the larger band and presence of a shorter product, which should correspond respectively to U3 precursor and to a product of degradation or misprocessing of the U3 snoRNA. Two bands are also detected for the snoRNA Tb9Cs3C3 and a single band for the snoRNA Tb10Cs4C3. * Indicates an unspecific hybridization. No clear difference was observed for the snoRNAs Tb9Cs3C3 and Tb10Cs4C3 between control and TbRRP44 deficient cells. (B) Analysis of…

• 

  Figure 10.. Overview of the pre-rRNA processing pathway in TbRRP44 deficient cells. (A) Diagram of T. brucei pre-rRNA structure with the positions of the cleavage sites. (B) Pre-rRNA intermediates of the SSU pathway. The 9.6 kb pre-rRNA is initially processed either at site B0 or B1 in ITS1 producing two SSU pre-rRNAs with different 3′-end, the longer identified as 3.5 kb (SSU-B1) and the shorter as 3.4 kb (SSU-B0). Subsequent non simultaneous cleavages of sites A0 and A1 liberate a 5′ ETS, producing up to four different species of the ∼2.5 kb SSU pre-rRNAs (A0-B1, A1-B1, A0-B0 and A1-B0). Based on the conservation of pre-rRNA processing in eukaryotes, it is very likely that TbRRP44 participates directly in the removal of part of the ITS1 segment that remains in the 3′ of the SSU precursors, which are finally processed at site A2, generating the mature 18S rRNA. (C) Tentative processing pathway of the 5.8 kb LSU-B0 pre-rRNA. Following cleavage of the 9.6 kb pre-rRNA at site B0, the LSU-B0 seems to be initially processed at site C2 separating the 0.7 pre-rRNA of the 5.8 rRNA pathway from the 5.1 kb pre-rRNA containing the remaining LSU pre-rRNAs. The 5.1 kb pre-RNA is processed at site G1 in…