ribose methyltransferase and SLA1 H/ACA snoRNA pseudouridi

37 Pages·2008·3.2 MB·English

MCB Accepts, published online ahead of print on 22 December 2008
Mol. Cell. Biol. doi:10.1128/MCB.01496-08
Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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Trypanosoma brucei spliced leader RNA maturation by the cap 1 2’-O-
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ribose methyltransferase and SLA1 H/ACA snoRNA pseudouridine
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synthase complex
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6 Jesse R. Zamudio,1,† Bidyottam Mittra,1 Arnab Chattopadhyay,2 James A. WohElschlegel,2
7 Nancy R. Sturm,1* and David A. Campbell1 T D
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8 Department of Microbiology, Immunology & Molecular Genetics1, Department of Biological n
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9 Chemistry2, David Geffen School of Medicine e
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10 University of California at Los Angeles, Los Angeles, CA 90095-1489, USA, m

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12 †Present Address: KCoch Institute, Massachusetts Institute of Technology, Cambridge, c
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13 Massachusetts 02139, USA m
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15 *Corresponding author: Department of Microbiology, Immunology & Molecular Genetics, J
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16 4825 Molecular Sciences Building, 609 Charles E. Young Drive East, University of California at a
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17 Los Angeles, Los Angeles, CA 90095-1489. Phone: [phone]. Fax: [phone]. E- 3
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18 mail: [email protected] 1
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20 running title: SL RNA processing complex t
21 word count M&M: 965
22 word count Intro/Results/Disc: 4417
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25 ABSTRACT
26 Kinetoplastid flagellates attach a 39-nt spliced leader (SL) upstream of protein-coding regions in
27 polycistronic RNA precursors through trans-splicing. SL modifications include cap 2’-O-ribose
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28 methylation of the first four nucleotides and pseudouridine (ψ) formation at uracil 28. In
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29 Trypanosoma brucei TbMTr1 performs 2’-O-ribose methylation of the first transcribed
30 nucleotide, or cap 1. We report the characterization of an SL RNA procTessing complex with D
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31 TbMTr1 and the SLA1 H/ACA small nucleolar riboucleprotein particle that guides SL ψ n
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32 formation. TbMTr1 is in a high molecular weight complex containing the four conserved core e
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33 proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated Methyltransferase m

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34 associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable, but have tp
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35 decreased SL RNA Cprocessing efficiency in cap methylation, 3’-end maturation and ψ c
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36 formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP, but m
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37 does not affect U1 small nuclear RNA methylation. A complex methylation profile in the /

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38 mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. J
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39 The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple r
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40 modifications on the SL RNA substrate. ,

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41 INTRODUCTION
42 Mature RNA molecules contain extensive post-transcriptional nucleotide modifications
43 on both base and ribose moieties. The isomerization of uracil to pseudouridine (5-ribosyluracil)
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44 and 2’-O-ribose methylation, occur by either site-specific (31, 50) or small nucleolar RNA
45 (snoRNA) guided enzymes (13). Conserved snoRNA-guided ribosomal RNA (rRNEA)
46 nucleotide modifications occur in functionally relevant regions (14), with their absence resulting
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47 in disease states due to defective catalytic RNA activity (76). Cap ribose methylations of RNA n
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48 polymerase II transcripts by cap-specific methyltransferases (MTases) are conserved in higher e
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49 eukaryotes and implicated in the enhancement of translational efficiency (52). o
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50 Kinetoplastid protozoa, including the human-pathogenic Leishmania major, tt
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51 Trypanosoma brucei and Trypanosoma cruzi, transcribe all protein-encoding genes
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52 polycistronically (28, 40). Maturation to translatable monocistronic units requires resolution of s
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53 each coding region by trans-splicing of a 39-nt spliced leader (SL) exon and 3’-end g
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54 polyadenylation. The SL RNA is involved in the maturation of each and every nuclear mRNA,
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55 accounting for approximately 7% of total RNA synthesis (8, 23). Rapid substrate SL a
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56 consumption argues for a dynamic processing mechanism. Substrate SL RNA is modified by 3
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57 eight methylations of the 5-nt cap structure and pseudouridylation at nucleotide 28 (ψ ). Along 1
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58 with the m7G (cap 0), the methylations of the kinetoplastid cap 4 are the most extensive, with 2’- g
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59 O-ribose methylation of the first four nucleotides and additional base methylations on the first t
60 (m 6A) and fourth (m3U) positions (5, 21, 49). The SL cap 4 and/or exon primary sequence and
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61 pseudouridylation have been implicated in kinetoplastid trans-splicing (6, 39, 62, 67) and
62 polysome association (82).
63 Among higher eukaryotes, co-transcriptional cap 0 formation on mRNA and small
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64 nuclear RNA (snRNA) is accompanied often by conserved cap 2’-O-ribose methylation of the
65 first and second transcribed nucleotides, known as cap 1 and cap 2 (22). TbMTr1 is the
66 sequence-specific cap 1 2’-O-ribose MTase acting on the SL RNA and U1 snRNA in T. brucei
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67 (44, 77). Two additional T. brucei cap 2’-O-ribose MTases are named for the nucleotide
68 positions modified: TbMTr2 and TbMTr3 (1, 2, 24, 78). Cap 4 formation occurs sEequentially
69 from the 5’ end (66), with co-transcriptional (37) and intracellular-trafficking-dependent (81) cap
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70 formation proposed. TbMTr2 and TbMTr3 activities are dependent on SL RNA association with n
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71 the heptameric Sm-protein complex in vivo (38, 61, 79). Knockout lines for each of the cap 2’- e
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72 O-ribose MTase are viable (1, 2, 77). o
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73 The majority of snoRNAs direct nucleotide modifications on snRNA and rRNA by base- tt
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74 pairing with substrate RNA molecules (41). Box C/D snoRNAs guide predominantly 2’-O-
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75 ribose methylation while box H/ACA snoRNAs specify pseudouridine formation; both classes s
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76 may direct the cleavage of pre-rRNA. The C/D and H/ACA snoRNAs are each associated with g
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77 four distinct subsets of core proteins, conserved from archaea to eukaryota (41). Dual-function
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78 small Cajal-body-specific RNAs guide methylation and pseudouridylation on snRNAs (11, 27). a
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79 The H/ACA ribonucleoprotein (RNP) pseudouridylation core proteins are Cbf5p (a.k.a. 3
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80 Dyskerin), Nop10p, Nhp2p, and Gar1p (72). Cbf5p is the pseudouridine synthase that is 1
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81 essential in S. cerevisiae (29) and T. brucei (6). Mutations in the mammalian H/ACA snoRNP
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82 core proteins Dyskerin, Nop10, or Nhp2 cause dyskeratosis congenita (9, 56, 70). snoRNA s
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83 complexes have other functions, such as the five C/D RNPs directing rRNA cleavage and the
84 mammalian H/ACA telomerase RNA that templates telomere extension (64). The cleavage
85 snoRNPs are associated specifically with additional proteins to tailor their functions (17, 35, 57),
86 however none of the pseudouridylation snoRNPs are known to complex with factors beyond the
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87 four core components.
88 The T. brucei H/ACA snoRNAs form a single stem-loop structure with an AGA motif
89 (32), in contrast to the two stem-loop structures with ACA motif observed in other eukaryotes.
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90 Thirty-four H/ACA RNAs are predicted to guide modification of 32 rRNA nucleotides in T.
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91 brucei (34). The SLA1 H/ACA snoRNA guides ψ formation (33) and resides primarily in the
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92 nucleolus (53), but is also detected in the nucleoplasm (33). Cbf5 knocTkdown in T. brucei D
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93 destabilizes SLA1 snoRNA, with defects seen in SL RNA cap 4 formation and decreased trans- n
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94 splicing (6). Pseudouridine formation is not required for trans-splicing (39, 62). e
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95 We report the in vivo purification of a specialized SLA1 snoRNP complex containing
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96 TbMTr1 for enhanced SL RNA processing. TbMTr1 tandem affinity purification followed by tp
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97 protein identificatioCn using Multidimensional Protein Identification Technology (MudPIT) (20, c
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98 36) and the T. brucei genome database (7, 25) revealed association with the four conserved core m
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99 proteins of H/ACA snoRNPs, and a kinetoplastid-specific protein designated Methyltransferase g
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100 associated protein (TbMTAP). The SLA1 snoRNA is specifically associated with the TbMTr1 J
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101 complex. The TbMTr1 complex establishes a SL RNA processing center for Sm protein- a
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102 independent modifications, and demonstrates specialization of a pseudouridylation snoRNP for 3
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103 enhanced processing of a specific RNA. 1
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105 MATERIALS AND METHODS t
106 DNA cloning. The pMOT3H-TbMTr1 vector for in situ C-terminal HA-fusion protein
107 expression was cloned using pMOT3H (45). The last 724 nt of TbMTr1 coding region was
108 released from a pTOPO-TA (Invitrogen) vector containing the full 1110-bp coding region (77)
109 using an internal ApaI and an XhoI site engineered into the reverse primer for amplification. The
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110 TbMTr1 3’ UTR was amplified with TbMT370-3’UTR-R-NotI (5’-ATC TAG ACA AGT CAT
111 ACC TAG TCC) and BamHI370-3’UTR-F (5’-AGG ATC CCA AGT CAT AGG TAG TCC)
112 and inserted using XbaI and BamHI sites. The pMOT3H-TbMTr1 vector was digested with ApaI
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113 and BamHI for transfection. For PTP tagging, the TbMTr1 sequence was amplified from the
114 pTOPO-TA-TbMTr1 vector using MT370 Xba-F (GGT CTA GAA TGC CTG CCEG TTG CAG
115 AC) and pC-PTP-Neo370R (TGC GGC CGC AAT GAC TTG ACC TC). The last 724 nt of the
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116 TbMTr1 ORF was then excised with ApaI and NotI and ligated to the pC-PTP-NEO vector (58). n
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117 The pC-PTP-NEO-TbMTr1 vector was linearized with BbsI for transfection. e
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118 The full 1230-nt genomic coding sequence of TbMTAP (Gene DB Tb11.01.8210) was o
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119 amplified using primers TbSLA1_ApaI_F (AGG GCC CAT GCC AGC AAA AC) and tt
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120 TbSLA1_NotI_R (GCG GCC GCG ACA TTG AAA ATA G). The TbMTAP knockout vectors
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121 were made using the pKO vector (30) where the drug marker was replaced with phleomycin or s
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122 puromycin. The 648-nt 5’ UTR directly upstream of the ORF was cloned using g
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123 TbSLA1_5’UTR_HindIII_F (AAA GCT TGA GAA TTT AGC ACA C) and TbSLA1_5’-
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124 UTR_EcoRI_R (TGA ATT CCT CTG ATT CCT GAT G). The 496-nt 3’ UTR was amplified a
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125 with TbSLA1_3’-UTR_Xba_F (ATC TAG AAA TAA TTT TTA AAG AAC AC) and 3
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126 TbSLA1_3’-UTR_NotI_R (GCG GCC GCT ACT CAC AAA AC). Both UTRs were cloned 1
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127 into pKOpuro and pKOphleo, sequenced, and digested with HindIII/NotI for transfection.
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128 T. brucei strains and cell culture. YTAT procyclic T. brucei was grown at 27°C in SM s
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129 medium (10) supplemented with 10% fetal bovine serum. Transfection was performed as
130 described (71), and selected with hygromycin (20 µg/ml), puromycin (10 µg/ml), phleomycin
131 (2.5 µg/ml) or G418 (15 µg/ml).
132 DNA and RNA analyses. Total cell RNA was isolated with TRIzol reagent (Invitrogen), and
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133 high-resolution acrylamide RNA blotting, RNA primer extension using Moloney murine
134 leukemia virus reverse transcriptase (RT), and DNA sequencing reactions were performed as
135 described (80). The [γ-32P]-labeled oligonucleotides are: exon specific TbWTexon (CAA TAT
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136 AGT ACA GAA ACT G); intron-specific SL RNA TbSL40 (CTA CTG GGA GCT TCT CAT
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137 AC); U1 snRNA TbU1-40 (CCC ACT CAA AGT TTA CTG); TbSLA1 snoRNA TbSLA (GTC
138 TCG CTC TCC AGT TCG TG); Tb9cs2H1 snoRNA Tb9cs2H1R (AATT TCT CGG ACC ACG D
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139 TGA); Tb6CS1H2 snoRNA Tb6CS1H2R (CGC GGG TCC GAT TGA G); and U3 snoRNA n
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140 TbU3 (CCG GGC GGA GCC AGC AAC CTT C). Poly(A)+ RNA was isolated using the e
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141 MicroPoly(A)Purist kit (Ambion). All blots were visualized on PhosphorImager screens
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142 (Amersham). tp
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143 Total RNA fCrom 5 x 108 T. brucei cells was used for pseudouridine detection by N- c
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144 cyclohexyl-N’-(2-morpholinoethyl)-carbodiimide metho-p-toluolsulfonate (CMC) (Sigma) or m
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145 hydrazine-aniline treatment as described (3). The RNA pellet was treated with 35% hydrazine g
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146 solution (Sigma) and resuspended in 1.0 M aniline (Sigma). J
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147 Sucrose gradient fractionation. Nuclear extracts were prepared essentially as described (15) a
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148 from 1 l culture of T. brucei grown to a density of 107 cells/ml. Recombinant TbMTr1 was 3
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149 expressed and purified from E. coli as described previously (44) and was subjected to sucrose 1
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150 gradient sedimentation simultaneously. Twenty serial fractions were collected from the top of g
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151 the gradients using a Piston Gradient Fractionator (BioComp Instruments), TCA precipitated, t
152 and subjected to SDS-PAGE followed by immunoblot analysis. Proteins standards (Amersham
153 Bioscience) soybean trypsin inhibitor (20.1 kDa), bovine serum albumin (67 kDa), lactate
154 dehydrogenase (140 kDa), catalase (232 kDa), and ferritin (440 kDa) were loaded on sucrose
155 gradients under similar conditions and detected by Coomassie staining after SDS-PAGE.
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156 Tandem affinity purification and mass spectrometry. Purification was performed with 2.5 l
157 of 1 x 107 cell/ml culture. For tandem affinity purification, the PTP tag with protein A and
158 human protein C epitope was utilized and purification performed as described (58). Total elution
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159 from Protein C column was resolved on SDS-PAGE and stained with GelCode Blue Stain
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160 Reagent (Thermo Scientific) or TCA precipitated. For analysis by mass spectrometry, TCA
161 precipitates were resolubilized in digestion buffer (8 M urea, 100 mM TTris-HCl pH 8.5) and D
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162 digested by the sequential addition of lys-C and trypsin proteases as described (42). Peptide n
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163 digests were analyzed using a 2D-LC-MS/MS approach in which they were fractionated using 7- e
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164 step multidimensional separation strategy and eluted directly into a Thermofisher LTQ-Orbitrap
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165 mass spectrometer where tandem mass spectra were collected in a data-dependent fashion. tp
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166 Additional technicalC details regarding this method can be found in MacCoss et al. and Florens et c
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167 al. (20, 36). The proteomic data was analyzed using the SEQUEST and DTASelect2 algorithms m
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168 (18, 63) against the T. brucei genome database (7, 25). The final filtering criteria used for this g
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169 analysis was a peptide-level false positive rate of 5% as estimated using a decoy database and a J
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170 minimum of two peptides per protein (48). a
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171 Immunoprecipitation and Western blotting. Cells from 150 ml of culture at 1 x 107 cells/ml 3
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172 were harvested, lysed as described in the PTP purification and 5 µg of HA.11 monoclonal
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173 antibody (Covance) added. After mixing gently for 1 h at 4°C, 50 µl of Protein G sepharose g
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174 suspension (G.E. Healthcare) was added and mixed for 1 h. The samples were spun at 12000 g t
175 at 4°C, the pellet washed 4 x in Buffer C (100 mM NaCl, 20 mM Tris-HCl pH 8.0, 3 mM MgCl ,
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176 0.1% Tween-20), and resuspended directly into SDS-PAGE loading buffer, or the RNA was
177 extracted with TRIzol reagent. Protein blots were probed with 1:1000 dilution of HA.11 αHA
178 monoclonal antibody (Covance), 1:1000 dilution of αHIS polyclonal antibody (Santa Cruz),
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179 1:1000 dilution of polyclonal rabbit αHA antibody (Santa Cruz) or 1:1000 dilution of peroxidase
180 anti-peroxidase agent (Fisher).
181
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182 RESULTS
183 In vivo TbMTr1 is in a higher molecular weight complex. TbMTr1 is the cap 1 E2’-O-ribose
184 MTase modifying the SL RNA and U1 snRNA in T. brucei (77). In seaTrch of additional proteins D
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185 involved in snRNA biogenesis, TbMTr1 protein interactions were queried. n
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186 Procyclic T. brucei cell line TbMTr1–/HA expressing a functional C-terminal e
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187 hemagglutinin (HA)-tagged TbMTr1 (TbMTr1-HA) was established. TbMTr1-GFP fusion
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188 protein localized in the nucleus (77), thus nuclear extracts from TbMTr1–/HA cells were ttp
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189 fractionated on a 2-2C0% sucrose gradient and compared to the sedimentation of purified c
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190 recombinant His-tagged TbMTr1 (rTbMTr1-His) expressed in E. coli. Fractions collected from m
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191 each gradient were assessed for the presence of TbMTr1 using antibodies recognizing the g
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192 appropriate epitope tags. Purified rTbMTr1-His peaked in fractions 1 and 2 between protein J
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193 standards of 20.1 kDa and 67 kDa (Figure 1A), consistent with a monomeric 41.9-kDa form. a
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194 TbMTr1-HA protein from T. brucei nuclear extracts sedimented faster, peaking in fraction 6, 3
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195 near the 232-kDa protein standard. A detectable amount of monomeric protein was observed in 1
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196 some experiments (see Figure 4C) and may reflect either small amounts of monomeric TbMTr1 g
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197 within T. brucei cells or disassociation of the complex under fractionation conditions. The t
198 absence of higher molecular weight rTbMTr1-His forms indicated that the in vivo complexes
199 were not multimeric oligomerization products, implying that in vivo TbMTr1-HA was associated
200 with additional proteins to form the faster sedimenting complex.
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201 To identify the interacting components, the TbMTr1 complex was isolated by tandem
202 affinity purification.
203 Purification of the TbMTr1 complex. Procyclic T. brucei cell line TbMTr1–/PTP was
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204 engineered to express a functional TbMTr1 C-terminal PTP tag fusion, which provides higher
205 yield and less contaminants than the original TAP epitopes (58). A slightly faster mEigrating
206 single band corresponding to the predicted 59.7-kDa TbMTr1-PTP protein was confirmed in
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207 whole cell extracts by detection of the protein A epitope with peroxidase anti-peroxidase reagent n
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208 (Figure 1B). Following purification, SDS-PAGE revealed five protein bands with apparent e
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209 molecular weights of 48, 46, 45, 28 and < 20 kDa in approximately stoichiometric ratios (Figure o
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210 1C). tt
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211 To identify the proteins in the final elution of the purification protocol, analysis of the
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212 complex peptide mixture was performed in solution using MudPIT and the T. brucei genomic s
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213 database. g
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214 TbMTr1 associates with core H/ACA snoRNA proteins. Applying a two-peptide hit per
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215 protein criterion for MudPIT analysis, the 105 total peptide sequences obtained corresponded to a
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216 six T. brucei proteins, summarized in Table 1. In addition to the TbMTr1-PTP bait protein, four 3
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217 conserved core protein components of H/ACA snoRNPs Cbf5p, Gar1p, Nhp2p, and Nop10p 1
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218 were identified, along with a hypothetical T. brucei ORF.
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219 Proteins identified by MudPIT had predicted sizes that were consistent with the SDS- s
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220 PAGE of the TbMTr1-PTP purification. The largest protein detected was the 48-kDa homolog
221 of the H/ACA snoRNA guided pseudouridine synthase Cbf5p. The 28-kDa and <20-kDa bands
222 matched homologs to known Cbf5p-associated proteins Gar1p and Nhp2p. The final core
223 component of H/ACA snoRNPs, Nop10p, with a predicted molecular weight of 7.4 kDa, was run
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