The messenger RNA decapping and recapping pathway in Trypanosoma

Anna V. Ignatochkinaa,1, Yuko Takagib,1,2, Yancheng Liuc, Kyosuke Nagataa, and C. Kiong Hoa,b,c,d,3

aDepartment of Infection Biology, Graduate School of Comprehensive Human Sciences and the cHuman Biology Program, University of Tsukuba, Ibaraki 305-8575, Japan; and Departments of bBiological Sciences and dMicrobiology and Immunology, State University of New York, Buffalo, NY 14260

Edited by Roy Parker, University of Colorado, Boulder, CO, and approved April 23, 2015 (received for review January 12, 2015)

The 5′ terminus of trypanosome mRNA is protected by a hyper- studies suggest that stable uncapped mRNAs accumulate in cells methylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma and that they can potentially acquire a 5′-cap structure to re- brucei nuclear capping with cap guanylyltransferase and generate translatable mRNAs (14, 15). However, the mRNAs that methyltransferase activities (TbCgm1) modifies the 5′-diphosphate are decapped or cleaved by endonucleases are produced as pRNAs RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show and thus need to be converted to diphosphorylated (ppRNAs) that T. brucei cytoplasmic (TbCe1) is a bifunctional to be capped by a guanylyltransferase. The existence of pRNA 5′-RNA and guanylyltransferase that transfers a γ-phosphate kinase in mammalian cells has been proposed (16, 17) and such from ATP to pRNA to form ppRNA, which is then capped by transfer an activity has been found in the cytosol, complexed with a gua- of GMP from GTP to the RNA β-phosphate. A Walker A-box motif in nylyltransferase (18). However, the responsible protein has not yet the N-terminal domain is essential for the RNA kinase activity and been identified. is targeted preferentially to a SL RNA sequence with a 5′-terminal In trypanosomes, cap 4 structure is derived from cap 0 and methylated nucleoside. Silencing of TbCe1 leads to accumulation formed exclusively on a 39-nucleotide spliced leader (SL) RNA, of uncapped mRNAs, consistent with selective capping of mRNA that which is transferred by trans-splicing to individual ORFs derived has undergone trans-splicing and decapping. We identify T. brucei from a polycistronic transcript (19, 20). The cap 0 is formed by mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped an RNA triphosphatase, TbCet1, and a bifunctional guanylyl- RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also -methyltransferase, TbCgm1 (21–23). In addition, three remove GDP from unmethylated capped RNA but is less active at a 2′-O-nucleoside RNA methyltransferases implicated in cap 4 meth- BIOCHEMISTRY mature cap 4 end and thus may function in RNA cap quality surveil- ylation on the SL RNA have been identified and characterized lance. Our results establish the enzymology and relevant protein cat- (24–27), and a stand-alone guanylyltransferase (TbCe1) and a alysts of a cytoplasmic recapping pathway that has broad implications stand-alone cap methyltransferase (TbCmt1) have been identi- for the functional reactivation of processed mRNA ends. fied (28, 29). Here we show that TbCe1 is a cytoplasmic recapping enzyme with ′ mRNA recapping | mRNA decapping | RNA repair | cap methylation | 5 -monophosphate RNA kinase and guanylyltransferase activities Trypanosoma brucei and that it can convert pRNA into GpppRNA via a ppRNA inter- mediate. We also identify TbDcp2 as the Nudix-family decapping enzyme that generates pRNA from a capped RNA. Our findings he earliest modification to the eukaryotic mRNA is the ad- define a decapping/recapping pathway in trypanosoma and its dition of a cap structure (m7GpppN; cap 0) at the 5′ end, to T responsiveness to cap methylation status. protect the mRNA from degradation and recruit factors that promote RNA splicing, export, and translation initiation (1). The cap is formed in the nucleus by three sequential enzymatic re- Significance actions: the 5′ triphosphate of the nascent mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; the diphosphate end is cap- The 5′ end of eukaryotic mRNA is capped and methylated to ped with GMP by RNA guanylyltransferase; and the GpppRNA is protect mRNA from degradation and enhance protein synthesis. methylated by (guanine N7) methyltransferase to form cap 0 (2). However, the cap can be removed from mRNA by decapping. We Nucleotides adjacent to the cap are typically methylated on the first identified a recapping enzyme with 5′-monophosphate RNA ki- and second nucleosides to form cap 1 and cap 2 structures, re- nase activity from trypanosome and provide evidence that dec- spectively. The most elaborate cap structure, called cap 4, is found apped transcripts can be recapped to regenerate translatable in Trypanosoma brucei and other kinetoplastid parasites and con- mRNA. The kinase activity is dependent on mRNA leader se- sists of a standard cap 0 with 2′-O methylations on the first four quence and is stimulated by hypermethylation found in the ribose sugars (AmAmCmUm), and additional base methylations trypanosome mRNA. We also identify a trypanosome decapping on the first adenine (m6,6A) and the fourth uracil (m3U) (3). enzyme that removes cap structure from the mRNA, but is less Although additional methylations have been shown to enhance active on hypermethylated capped mRNA. These results suggest translation efficiency (4, 5), whether they affect the RNA decay that hypermethylated cap structure can influence certain tran- process is unknown. scripts to be preferentially decapped or recapped during the All mRNA is subject to degradation by either a 5′-to-3′ or a parasite life cycle. 3′-to-5′ exonucleolytic pathway, generally initiated by shortening of the poly(A) tail (6). In the 5′-to-3′ pathway, the cap is removed Author contributions: A.V.I., Y.T., and C.K.H. designed research; A.V.I., Y.T., Y.L., and C.K.H. as m7Gpp by the RNA decapping enzyme Dcp2, a member of the performed research; K.N. contributed new reagents/analytic tools; A.V.I., Y.T., and C.K.H. analyzed data; and C.K.H. wrote the paper. Nudix superfamily, leaving a 5′-monophosphorylated RNA (pRNA) (7, 8). The exposed pRNA is progressively de- The authors declare no conflict of interest. graded by a 5′-to-3′ exonuclease (Xrn1/Rat1). Incompletely cap- This article is a PNAS Direct Submission. ped RNAs that lack the N7 methyl moiety, as well as defective 1A.V.I. and Y.T. contributed equally to this work. mRNAs with premature termination codons, are decapped by a 2Present address: National Institute of Advanced Industrial Science and Technology, 1-1-1 cellular quality-control machinery (9–12). Higashi, Tsukuba, Ibaraki 305-8566, Japan. Studies in yeast and mammals suggest that decapping is a reg- 3To whom correspondence should be addressed. Email: [email protected]. ulated process, and the uncapped mRNAs can be sequestered from This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. degradation in the P bodies and stress granules (6, 13). Several 1073/pnas.1424909112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1424909112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Results radiolabeled γ-phosphate from ATP to both the chemically phos- TbCe1 Is a Cytoplasmic Capping Enzyme with a 5′-Monophosphate RNA phorylated 21-mer pRNA and enzymatically phosphorylated B Kinase Activity. TbCgm1 is deemed responsible for cap 4 bio- 24-mer pRNA but not to the pppRNA or OHRNA (Fig. 1 ). synthesis on the SL RNA, insofar as silencing of TbCgm1 increases Moreover, it converted the pRNA into a capped RNA in an ATP-dependent manner, as evidenced by radiolabel transfer of the abundance of uncapped SL RNA and leads to accumulation of α 32 α 32 C hypomethylated SL RNAs (22, 23). In contrast, silencing of TbCe1 [ - P]GMP from [ - P]GTP in the presence of ATP (Fig. 1 ). does not prevent SL RNA capping (23). To assess the function of Alanine mutations within the conserved nucleotide-binding TbCe1, we examined the subcellular localization of both TbCgm1 motif of TbCe1 (K49A, S50A, or K49A/S50A double mutations) and TbCe1 in T. brucei procyclic cells by indirect immunofluores- abolished its activity without affecting its gua- nylyltransferase activity (Fig. 2 A–D). We verified that the product cence with a protein A antibody that recognizes the PTP fusion ′ protein epitope tag (Fig. 1A and Fig. S1A). As expected, TbCgm1– generated by TbCe1 has a 5 -diphosphate end by isolating the radiolabeled RNA product from the gel and subjecting it to PTP localized to the nucleus, where SL RNAs are synthesized, nuclease P1 digestion (Fig. 2E). A small percentage (∼15%) of consistent with the finding that TbCgm1 is recruited to the SL RNA the radiolabeled product migrating at the position of GpppA promoter to cap the SL RNA (22). In contrast, TbCe1 localized to was observed, suggesting that the ppRNA formed by TbCe1 is the cytoplasm. We conclude that TbCe1 is a cytosolic enzyme and subsequently converted to GpppRNA due to the presence of likely functions in a different capping pathway from TbCgm1. preguanylated enzyme in the recombinant protein preparation. In- To evaluate whether phosphotransferase activity is intrinsic to deed, when GTP was included in the reaction, the ppRNA was TbCe1, recombinant protein was produced in bacteria and puri- – converted to a capped RNA, as evident from an increase in the fied from soluble lysates by nickel agarose and DEAE chroma- ratio of GpppA/ADP (Fig. 2E, lane 4). Notably, GpppA was not tography (Fig. S1B). TbCe1 was incubated with either a pRNA, a – ′ ′ detected when the TbCe1 K288A mutant protein (lacking the 5 -triphosphate-terminated SL RNA (pppRNA), or a 5 -hydroxyl- lysine nucleophile for enzyme–GMP formation) was tested (Fig. terminated RNA (OHRNA), with a sequence that corresponds E γ 32 2 , lanes 5 and 6). We conclude that TbCe1 is a bifunctional to the first 21 or 24 nt of SL RNA, in the presence of [ - P]ATP capping enzyme with novel 5′-monophosphate RNA kinase and and magnesium. TbCe1 was capable of effectively transferring guanylyltransferase activities that can convert pRNA into GpppRNA. The nucleotide-binding motif in the N-terminal domain of TbCe1 is essential for the RNA kinase activity, and is likely to be the that coordinates the ATP.

Characterization of pRNA Kinase Activity. The pRNA kinase activity was proportional to the TbCe1 concentration (Fig. 2D). We cal- culated a specific activity of 11 fmol of 32P-labeled RNA formed per nanogram of TbCe1 during a 15-min reaction, which corre- − sponds to a turnover number of 0.05 min 1. ATP and dATP were able to compete as phosphate donors, whereas other rNTP or dNTPs were not (Fig. 2F). Addition of GTP to the RNA kinase reaction shifted the radiolabeled RNA product by a single nu- cleotide, consistent with the formation of capped GpppRNA. The extent of pRNA phosphorylation was proportional to the ATP concentration and leveled off at 20 μMATP(Fig.2G). A Km of 2.7 μM ATP was calculated from nonlinear regression fitting of the data to the Michaelis–Menten equation using Prism. No RNA kinase activity was detectable in the absence of a divalent cation (Fig. 2H). Magnesium was a more effective than man- ganese, with an optimum of 0.2–0.5 mM MgCl2. The properties of the TbCe1 guanylyltransferase are similar to those of the TbCgm1 and RNA guanylyltransferases found in other eukaryotes and viruses (Fig. S2). TbCe1 was unable to transfer GMP to a triphosphate-terminated poly(A) unless the 5′ end was converted to a diphosphate end by RNA triphosphatase (Fig. S2E). The N-terminal 5′-monophosphate kinase activity is not essential for the C-terminal guanylyltransferase activity, as neither mu- tation abolishing the kinase activity (Fig. 2B) nor a deletion of the entire N-terminal kinase domain (28) affected the enzyme– GMP complex formation.

Fig. 1. TbCe1 is a cytoplasmic capping enzyme that possesses 5′-mono- The pRNA Kinase Activity Is Specific to the SL RNA and Stimulated by phosphate RNA kinase and guanylyltransferase activities. (A) Localization of Cap 4 Methylation. In trypanosomes, mRNAs acquire an identical PTP-tagged TbCgm1 and TbCe1. Procyclic trypanosomes expressing either 39-nt SL RNA through trans-splicing. The sequence of the pRNA TbCgm1–PTP (Top)orTbCe1–PTP (Bottom) were fixed and permeabilized, and substrate used to characterize the pRNA kinase and capping ac- the PTP–tag was detected by immunofluorescence. The kinetoplast and nu- tivity in this study corresponds to the first 21 nucleotides of the SL clear DNA were counterstained with DAPI. A typical image is shown. (B)TbCe1 RNA. To address whether this sequence influences the pRNA specifically phosphorylates pRNA. Reaction mixtures (10 μL) containing 50 mM · μ γ 32 kinase activity, we prepared a pRNA that has a sequence corre- Tris HCl (pH 8), 100 M[ - P] ATP, 1 mM DTT, 0.5 mM MgCl2,andeither2pmol + + – of 21-mer pppRNA, 21-mer pRNA, 24-mer RNA ,or RNA treated with sponding to nucleotides 8to 28 of the SL RNA [SL-pRNA(8 OH 24 OH 24 28)] and a 24-mer non-SL RNA (non-SL pRNA) (Fig. 3A). As polynucleotide kinase (pRNA24), as indicated, were incubated with 20, 100, B – or 500 ng of TbCe1 (proceeding from Left to Right within each titration shown in Fig. 3 , SL pRNA(8 28) and non-SL pRNA were less series). A control reaction lacking enzyme (−) is shown in the lane indicated. effective substrates than the SL pRNA, resulting in reductions to (C) RNA capping activity. Reaction mixtures (10 μL) containing 50 mM 9% and 0.8%, respectively, in specific activity compared with that 32 Tris·HCl (pH 8.5), 1 mM DTT, 0.5 mM MgCl2,20μM[α- P] GTP, 100 nM of when the SL pRNA was used. Substitution of A with G at po- pRNA, with TbCe1 and ATP concentration as specified. Position of the capped- sition 1 did not affect the kinase activity, whereas a substitution labeled 21-mer RNA is indicated. Radiolabeled phosphate is indicated in red. with either C or U led to a drop to 1% and 0.3% of the activity

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1424909112 Ignatochkina et al. Downloaded by guest on September 29, 2021 and SL pRNAr1234 were phosphorylated to a similar extent. The effect of 2′-O methylation on the TbCe1 kinase activity was further evaluated at various ATP concentrations (Fig. 4C). The stimulation was most pronounced at limiting ATP concentra- tions; in this context the increase in activity was 10-fold. The Km for ATP in the presence of the SL pRNAr1 and SL pRNAr1234 substrates was 0.26 μM and 0.36 μM, respectively; compared with 2.7 μM for the unmethylated SL pRNA. These results suggest that TbCe1 preferentially targets methylated RNAs derived from the mature cap 4 mRNA for recapping. Because cap 4 methyl- ation occurs in the nucleus and depends on the cap 0 structure, the hypermethylated uncapped pRNA must be derived from a mature mRNA that has undergone decapping.

TbCe1 Knockdown Accumulates Uncapped mRNAs. Basedontheabove- described experiments, we predicted that silencing of TbCe1 would prevent the recapping of certain mRNA transcripts and lead to the accumulation of pRNAs in the cell. To test this prediction, we performed a ligation-mediated RT-PCR assay to detect pRNAs in T. brucei (Fig. S3). The anchor RNA oligonucleotide was ligated with poly(A) RNA isolated from TbCe1 RNAi-induced and unin- duced cells, in the presence or absence of splint DNA, whose 3′ half is complementary to the anchor RNA sequence and whose 5′ half is complementary to the SL RNA sequence (Fig. 5 and Fig. S3). The RNA was converted to cDNA using oligo(dT) as a primer, and splint ligation products were detected by PCR using oligo(dT) and a DNA primer specific to the anchor sequence. A series of PCR products, in the range of 0.4–1.2 kb, was detected in TbCe1 RNAi cell lines (Fig. 5). The amounts of splint ligation products, as well as BIOCHEMISTRY their heterogeneity, were significantly higher in TbCe1-silenced Fig. 2. Characterization of TbCe1 pRNA kinase activity. (A) Mutational than the unsilenced control. Omission of splint DNA in the analysis. Aliquots (4 μg) of wild-type (WT) TbCe1 and the indicated Ala mu- parallel reaction resulted in loss of the majority of the signal, tants were subjected to SDS/PAGE and visualized with Coomassie blue. The positions of marker proteins (in kilodaltons) are indicated. (B) Guanylyl- transferase activity. Reaction mixtures (20 μL) containing 50 mM Tris·HCl (pH 32 8), 1 mM DTT, 1 mM MgCl2,25μM[α- P] GTP, and 100 ng of WT or mutant TbCe1 protein, as indicated in A, were incubated at 30 °C for 15 min. Enzyme– GMP complex (EpG) is indicated by an arrow. (C) RNA kinase activity. Reaction mixtures (20 μL) contained 50 mM Tris·HCl (pH 8), 20 μM[γ-32P] ATP, 1 mM

DTT, 1 mM MgCl2, and 100 nM of pRNA, and were incubated at 30 °C for 15 min. (D) Protein titration. Standard RNA kinase assay (10 μL) with indicated amount of WT or mutant TbCe1 protein. The extent of 32P-labeled RNA formed is plotted as a function of input enzyme. (E) Analysis of a reaction product. The 32P-labeled RNA products generated by WT and K288A in C were excised from the gel, digested with nuclease P1, and analyzed by TLC developed by 1 M formic acid and 0.5 M LiCl. (F) Competition assay. A re- action mixture (20 μL) containing 50 mM Tris·HCl (pH 8), 20 μM[γ-32P] ATP,

1 mM DTT, 1 mM MgCl2, and 100 nM of the SL pRNA, 50 ng of TbCe1, and 200 μM of the indicated nucleotides was incubated at 30 °C for 15 min. A control reaction without added competitor nucleotide is indicated (−). Asterisk indicates position of capped 21-mer RNA. (G) ATP dependency. The standard RNA kinase assay (10 μL) contained 200 ng of TbCe1 and the indicated con- centration of [γ-32P] ATP. The yield of 32P-labeled RNA was plotted as a func- tion of ATP concentration. (H) Metal dependency. Standard RNA kinase assay 32 contained 200 ng of TbCe1 and either MgCl2 or MnCl2. The yield of P-labeled RNA was plotted as a function of divalent cation concentration. The data shown represent the average of three separate experiments, with SE bars.

relative to that for the wild-type SL pRNA (Fig. 3B). These results demonstrate that TbCe1 preferentially targets SL RNAs and that the presence of a purine at position 1 is critical for its pRNA kinase activity. The findings that the TbCe1 RNA kinase activity is dependent on the SL RNA sequence prompted us to examine whether 2′-O methylations within the first 4 nucleosides can influence the ac- Fig. 3. Phosphotransferase activity is stimulated by spliced leader RNA se- tivity. We evaluated two methylated SL pRNAs for their effective- ′ quence. (A) The sequences of pRNA substrates are illustrated. (B) Specificity ness as substrates for TbCe1: one has 2 -O methylation at position for the SL RNA. Standard RNA kinase assay (10 μL) contained either 1 pmol of ′ A1 (SL pRNAr1), and another has 2 -O methylations at positions SL-pRNA, SL-pRNA(8–28), non-SL pRNA24, SL-pRNA(A1G), SL-pRNA(A1C), or A1, A2, C3, and U4 (SL pRNAr1234). The 2′-O methylation of the SL-pRNA(A1U), with either 20, 100, or 500 ng of TbCe1 (proceeding from pRNA enhanced the TbCe1 kinase activity ∼3-fold over that for a Left to Right within each titration series). A phosphorimager scan of the gel pRNA lacking any modification (Fig. 4 A and B). Both SL pRNAr1 is shown.

Ignatochkina et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 The RNA dependence was also demonstrated by competition assay using cap analogs (Fig. 6E); none of the cap analogs or GTP ef- fectively competed against the m7GpppRNA43 substrate. A 10-fold excess of hydroxyl-terminated RNA (1 μM) had a minimal effect on decapping activity, whereas a 100-fold excess of hydroxyl-termi- nated RNA (10 μM) resulted in 70% inhibition, suggesting that TbDcp2 recognizes both the cap and the RNA chain (Fig. 6E). In summary, our data demonstrate that TbDcp2 possesses canonical RNA decapping activity in vitro. Fig. 4. Effect of cap 4 methylation on TbCe1 phosphotransferase activity. (A) Protein titration. The standard RNA kinase assay (10 μL) contained TbDcp2 Preferentially Hydrolyzes Prematurely Hypomethylated RNA. 100 μM[γ-32P] ATP and 100 nM of either SL-pRNA, pRNAr1 (with 2′-O ribose To address whether cap 4 methylation can influence TbDcp2 methylation at position 1) or pRNAr1,2,3,4 (with 2′-O ribose methylation at activity, we prepared a cap-labeled SL RNA that resembles cap 1 position 1, 2, 3, and 4) and TbCe1, as indicated. The yield of 32P-labeled RNA (m7GpppRNAr1) and cap 4 (m7GpppRNAr1234) from the 21-mer was plotted as a function of input TbCe1. (B) Kinetics. Standard RNA kinase SL pRNA, using recombinant TbCe1 (SI Materials and Methods). assay (80 μL) contained 160 ng of TbCe1 and 100 nM of either SL-pRNA, The rate of m7Gpp release from the cap 1 substrate was pRNAr1, or pRNAr1,2,3,4. An aliquot (10 μL) was withdrawn at the indicated comparable to that of the cap 0 (Fig. 6F), indicating that 2′-O time point, and the products were resolved by denaturing PAGE. The yield of 32 methylation at position 1 does not affect decapping. In contrast, P-labeled RNA product is plotted as a function of incubation time. (C) ATP the rate of m7Gpp release from cap 4 was reduced ∼10-fold com- titration. The standard RNA kinase assay (10 μL) contained 200 ng of TbCe1, 100 nM of either SL-pRNA, pRNAr1, or pRNAr1,2,3,4, and [γ-32P] ATP at the pared with that of the cap 0, indicating that hypermethylated RNA indicated concentration. The yield of 32P-labeled RNA was plotted as a was resistant to decapping by TbDcp2. function of ATP concentration. The data shown represent the average of We also examined the effect of unmethylated capped SL RNA three separate experiments. SE bars are included for each datum point. (GpppRNA) on decapping activity. TbDcp2 can efficiently hy- drolyze Gpp from Gpp-terminated SL RNA (Fig. 6G). The rate of Gpp release was 40% faster than that of m7Gpp. Similarly, 2′-O although trace amounts of nonspecific ligation products were methylation at position 1 did not have significant influence on Gpp detected. These results suggest that decapped 5′-monophosphate- hydrolysis. However, the rate of decapping from GpppRNAr1234 terminated mRNA is the substrate for TbCe1 in vivo. Although was reduced by 65% compared with that for GpppRNA, consistent the trypanosome scavenger decapping enzyme DcpS, which hy- with the notion that hypermethylated RNAs are less efficiently drolyzes the m7GpppN cap dinucleotide, has been characterized decapped by TbDcp2. The optimal substrates for decapping by (30), a decapping enzyme that releases m7Gpp and generates a TbDcp2 were: GpppRNA ≥ GpppRNAr1 = m7GpppRNA ≥ pRNA has not yet been identified in this organism. m7GpppRNAr1 > GpppRNAr1234 > m7GpppRNAr1234. These results demonstrate that cap 4 methylation can influence the Identification of the T. brucei Decapping Enzyme. The cytosolic Dcp2 decapping efficiency of TbDcp2 and raise the prospect that decapping from fungi and metazoans belong to the Nudix TbDcp2 may function as a surveillance enzyme to decap mRNAs superfamily of (31), which release m7Gpp from capped lacking either N7 guanine methylation or ribose 2′O-methylation mRNA, generating pRNA. Five putative Nudix proteins have at nucleosides 2, 3, and 4. been identified by bioinformatics analysis of the T. brucei pro- teome: TbNudix1 (GenBank accession: EU711412.1), TbNudix2 (Tb927.5.4350), TbNudix3 (Tb11.01.1570), TbNudix4 (Tb927.6.2670), and TbNudix5 (Tb10.70.2530) (Fig. 6A). Among these proteins, TbNudix1 has been characterized as mitochondrial edited mRNA stability factor (32), and thus we did not test it for a decapping activity. TbNudix2 and TbNudix3 were found in the glycosome but their catalytic activities have not been defined (33). The functions of TbNudix4 and TbNudix5 are unknown. To determine if TbNudix2, TbNudix3, TbNudix4, and TbNudix5 possess an RNA decapping activity, we produced His-tagged fusion proteins in bacteria (Fig. 6B) and assayed for the release of m7Gpp in vitro using a cap-labeled 43-mer RNA (m7GpppRNA43). Lib- eration of m7Gpp was detected by TLC analysis. TbNudix4 was the only enzyme that led to release m7Gpp from m7GpppRNA43 in the presence of magnesium (Fig. 6C and Fig. S4). We verified that the product generated is m7Gpp by subjecting the reaction product to alkaline phosphatase digestion (Fig. S4). We therefore rename TbNudix4 as TbDcp2. The amino acid sequence of the 294 amino acid TbDcp2 is shown in Fig. S5A. TbDcp2 lacks a conserved amino-terminal Box A do- main and a carboxy-domain present in other Dcp2 proteins. TbDcp2 sedimented as a single peak, between BSA and cytochrome C in a parallel gradient, which suggested that it is a monomer in solu- B tion (Fig. S5 ). Its activity profile coincided with the distribution ′ of the TbDcp2 polypeptide (Fig. S5C), and we calculated a Fig. 5. TbCe1 knockdown results in accumulation of 5 -monophosphate RNA. Poly(A) RNAs from TbCe1 RNAi uninduced (−) and induced with tetracy- specific activity of 60 fmol of m7Gpp released per nanogram in −1 cline for the number of days indicated were subjected to ligation-mediated 1 min, which corresponded to a turnover rate of 2 min . RT-PCR (Fig. S3). Splint-ligation products were detected by PCR using an anchor What distinguishes decapping enzyme from nucleotide pyro- forward primer and oligo(dT) as a reverse primer (Top). Products were re- phosphatase is a dependence on RNA. TbDcp2 hydrolyzed m7Gpp solved on agarose gel. Control reactions in which the splint DNA was omitted from 33-mer and 23-mer RNA with an efficiency similar to that for were carried out in parallel. Internal control reaction was performed using the 43-mer substrate (Fig. 6D), but the activity was significantly SL-specific forward primer and an α-tubulin-specific reverse primer (Bottom). reduced with the 13 mer (∼30% compared to m7GpppRNA43). The positions of marker DNA (in kilobases) are indicated to the Right.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1424909112 Ignatochkina et al. Downloaded by guest on September 29, 2021 Fig. 6. Identification and characterization of T.bru- cei Nudix protein with RNA decapping activity. (A) Alignment of Nudix motif from five Trypanosoma brucei Nudix proteins (TbNudix), the yeast, fly, and human Dcp2 proteins, and the human Nud16 pro- tein. (B) Protein purification. The TbNudix proteins

were expressed and purified as a His10–tag fusions. A Coomassie blue-stained gel is shown. (C) Decapping activity. The standard decapping assay mixture (10 μL) 32 contained 100 nM of P cap-labeled m7GpppRNA43 and 20 ng of indicated TbNudix proteins. A phos- phorimager scan of the TLC plate is shown. (D)RNA dependency. The standard decapping assay mixture (20 μL) contained 2 ng of TbNudix4 and 100 nM of cap- labeled RNAs as indicated. Liberation of m7Gpp is plotted as a function of incubation time. (E) Com- petition assay. Standard decapping assay contained 32 100 nM of P cap-labeled m7GpppRNA43, 1 ng of TbNudix4 with either 10 μM of the indicated cap dinucleotide analog, 10 μM GTP, 0.1 μM, or 1 μMof

m7GpppRNA43, and 1 μMor10μMoftheOHRNA. A control reaction (none) was incubated in parallel without any competitor. Decapping activity was normalized to that of the control reaction without competitor (defined as 1.0). (F) Effect of 2′-O methylation on decapping activity. Standard decapping assay mixture (30 μL) containing 15 ng of TbNudix4, with 100 nM of 32P-labeled cap 0 (m7GpppRNA), cap 1 (m7GpppRNAr1), or cap 4 (m7GpppRNAr1234). Liberation of m7Gpp is plotted as a function of incubation time. (G) Identical to F except that the substrates for the assay were Gppp-RNA, Gppp-RNAr1, or Gppp-RNAr1,2,3,4. The data shown represent the average of three separate experiments. SE bars are included for each datum point.

Discussion We speculate that TbCmt1, a second guanine-N7 methyltransfer- TbCe1 Kinase Activity Is Preferentially Targeted to Hypermethylated ase encoded by trypanosomes, functions in the recapping pathway BIOCHEMISTRY Uncapped SL RNAs. In this report, we present the first evidence to to convert the TbCe1-generated GpppRNA into a translatable our knowledge that a 5′-monophospate RNA kinase activity is m7GpppRNA (29). Indeed, silencing of TbCmt1 did not affect the directly involved in the recapping of pRNA. The kinase and the growth of procyclic cells or capping of the SL RNA, similar to the guanylyltransferase activities are physically linked as a single phenotype observed in TbCe1 depletion (23). TbCe1 polypeptide, and TbCe1 depletion results in an accu- mulation of pRNA in T. brucei. The fact that the RNA kinase TbDcp2 Is RNA Decapping and Surveillance Enzyme. Of the four T. brucei activity depended on the SL RNA sequence and was stimulated Nudix proteins characterized, TbDcp2 (TbNudix4) is by methylation of cap 4 provides strong evidence that this en- the only one capable of releasing m7Gpp from m7GpppRNA in zyme preferentially acts on uncapped hypermethylated mRNAs a magnesium-dependent manner. Cap analogs were not effective that have undergone decapping. Because cap 2′-O ribose meth- competitors for TbDcp2, but uncapped RNA effectively inhibited yltransferases are in the nucleus but decapping likely takes place Dcp2 activity, a property shared by other RNA decapping enzymes in the cytoplasm, uncapped hypermethylated pRNAs with a 5′ SL (8, 36, 37). TbDcp2 may also function as a surveillance enzyme that would most likely accumulate in the cytoplasm, where TbCe1 removes incompletely capped mRNA, consistent with the decapp- resides. This substrate specificity of TbCe1 would likely exclude ing activity detected in cytoplasmic extracts from Leptomonas sey- capping of other RNA types (such as tRNAs, rRNAs, and non- mouri (37). Our finding that TbDcp2 activity is influenced by cap 4 coding RNAs) that are present at high levels in the cytoplasm. methylation suggests that the 5′-to-3′ turnover may be regulated by The endonucleolytically cleaved RNAs are also likely to be ex- differential cap methylation. cluded for recapping by TbCe1, thereby preventing the genera- tion of N-terminally truncated proteins, a scenario that could be Role of Cap 4 Modification in the Decapping and Recapping Pathway. potentially deleterious to the parasite. Hypermethylated cap 4 is a unique feature of the mRNA of The pRNA kinase activity of TbCe1 required the Walker A kinetoplastids and is involved in trans-splicing and translational nucleotide binding motif within the N-terminal region. The enhancement (5, 22). Although cap 4 methylation partially protects phosphotransferase activity is likely to be mechanistically similar mRNA from decapping by TbDcp2, the decapped transcripts— ′ — to the reaction carried out by and 5′-OHRNA which likely retain the methyl moieties on their 5 end may have , given that the following two residues were essential for an innate stability, inhibiting 5′-to-3′ degradation by exonuclease. If this activity (34, 35): invariant Lys49, which in the other kinases so, stable uncapped methylated pRNA may accumulate in the stabilizes the phosphate groups on the NTP, and conserved cytoplasm. Unless it is degraded by the 3′-to-5′ decay pathway, Ser50, which likely coordinates the divalent cation for phosphoryl TbCe1 can preferentially recap the hypermethylated pRNA. transfer. The fact that either ATP or dATP can serve as a phos- phate donor, whereas other rNTPs and dNTPs did not complete Model of mRNA Decapping and Recapping Pathway in Trypanosomes. with ATP, implied that TbCe1 recognizes the adenine base. The Fig. 7 summarizes a proposed model for mRNA decapping and enzymatic properties of TbCe1 are similar to those of the 5′-RNA recapping pathway in trypanosomes. In the nucleus, the SL RNA is kinase purified from crude fractions prepared from vaccinia virions capped by TbCet1 and TbCgm1 and then hypermethylated by a with respect to their magnesium and RNA dependence, as well as series of cap 4 methyltransferases. Trans-spliced and polyadenylated their nucleotide (ATP or dATP) specificity as a phosphate donor mature mRNAs are transported to the cytoplasm for protein syn- (17). However, TbCe1 does not appear to convert ppRNA to thesis. TbDcp2 may act as a conventional RNA decapping enzyme pppRNA. Further mutational and structural analysis is expected to to generate uncapped pRNAs. It may also decap mRNA with un- provide insights into how TbCe1 interacts with the SL sequence and methylated or hypomethylated cap structures. The decapped how its phosphotransferase activity is enhanced by cap 4 methylation. mRNAs are either digested by 5′-to-3′ exonuclease or sequestered In order for a translatable mRNA to be generated through re- from the degradation machinery and stored in a cytoplasmic com- capping, the GpppRNA must be methylated at the N7 position. partment. Under certain conditions, the recapping apparatus may

Ignatochkina et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 is resolved by trans-splicing and ; (iii)themature mRNA is transported to the cytoplasm; (iv) unwanted mRNAs are selectively degraded; and (v) the mRNA to be translated is protected. Overall, this requires more time and resources than the expression of a single gene in other eukaryotes. The decapping and recapping pathway may function to regulate the gene expression in response to stress or sudden environmental changes. Materials and Methods Two critical protocols are briefly described below and more details can be found in SI Materials and Methods.

RNA Kinase Assay. The standard reaction contained 50 mM Tris·HCl (pH 9.0), Fig. 7. Proposed model of mRNA decapping and recapping in T. brucei.Na- 32 1 mM DTT, 0.5 mM MgCl2,100μM[γ- P] ATP, 100 nM 5′-monophosphorylated ′ scent SL RNA with 5 triphosphate end is converted to diphosphate by RNA RNA, and TbCe1 protein for 15 min at 30 °C, unless otherwise specified. triphosphatase (TbCet1), capped and methylated by a nuclear capping enzyme Products were resolved by 18% (wt/vol) Urea-PAGE, and radiolabeled (TbCgm1), and hypermethylated by a series of cap 4 methyltransferases (col- product was visualized and quantitated by phosphorimager. lectively indicated as cap 4 MTases). Trans-spliced and polyadenylated cap 4 mRNA can enter the translational pool, whereas unmethylated capped and RNA Decapping Assay. The standard reaction, containing 50 mM of Tris·HCl hypomethylated capped mRNA are subject to either 3′-to-5′ degradation (not shown) or converted into pRNA by a decapping enzyme (TbDcp2). The dec- (pH 7.5), 2 mM MgCl2, 100 nM of cap-labeled RNA substrate and protein as apped pRNA can either be degraded by a 5′-3′ exonuclease, or have its 5′ cap specified, was incubated for 10 min at 25 °C. EDTA was added to a final restored by TbCe1 and TbCmt1 and then return to the translational pool. concentration of 16 mM to quench the reaction. An aliquot of the reaction mixture was spotted onto TLC PEI Cellulose F (Millipore EMD), and was de- veloped with 0.75 M LiCl. Liberation of the radiolabeled 5′ terminus was restore the uncapped pRNA to GpppRNA via TbCe1 and meth- visualized and quantified by phosphorimager analysis. ylate the N7 moiety of the cap via the TbCmt1, to regenerate translatable mRNA. ACKNOWLEDGMENTS. We thank Laurie Read (SUNY Buffalo) for T. brucei The mRNA recapping pathway is particularly advantageous DNA and Chris Lima (Sloan-Kettering Institute) for the expression vector. for trypanosomes and other kinetoplastid organisms that rely on We also thank Beate Schwer (Cornell University), Stewart Shuman (Sloan- trans Kettering Institute), and members of the Laboratory of Infection Biology a -splicing mechanism for gene expression. In trypanosomes, (University of Tsukuba) for their helpful suggestions. This material is the selective expression of a single gene of interest requires that based upon work supported by the National Science Foundation under (i) a long polycistronic pre-mRNA is transcribed; (ii) the transcript Grant 1050984.

1. Furuichi Y, Shatkin AJ (2000) Viral and cellular mRNA capping: Past and prospects. Adv 20. Liang XH, Haritan A, Uliel S, Michaeli S (2003) trans and cis splicing in trypanoso- Virus Res 55:135–184. matids: Mechanism, factors, and regulation. Eukaryot Cell 2(5):830–840. 2. Shuman S (2001) Structure, mechanism, and evolution of the mRNA capping appa- 21. Ho CK, Shuman S (2001) Trypanosoma brucei RNA triphosphatase. Antiprotozoal ratus. Prog Nucleic Acid Res Mol Biol 66:1–40. drug target and guide to eukaryotic phylogeny. J Biol Chem 276(49):46182–46186. 3. Bangs JD, Crain PF, Hashizume T, McCloskey JA, Boothroyd JC (1992) Mass spec- 22. Ruan J-P, Shen S, Ullu E, Tschudi C (2007) Evidence for a capping enzyme with spec- trometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J Biol ificity for the trypanosome spliced leader RNA. Mol Biochem Parasitol 156(2):246–254. Chem 267(14):9805–9815. 23. Takagi Y, Sindkar S, Ekonomidis D, Hall MP, Ho CK (2007) Trypanosoma brucei en- 4. Kuge H, Richter JD (1995) Cytoplasmic 3′ poly(A) addition induces 5′ cap ribose codes a bifunctional capping enzyme essential for cap 4 formation on the spliced methylation: Implications for translational control of maternal mRNA. EMBO J 14(24): leader RNA. J Biol Chem 282(22):15995–16005. 6301–6310. 24. Arhin GK, Ullu E, Tschudi C (2006) 2′-O-methylation of position 2 of the trypanosome 5. Zamudio JR, Mittra B, Campbell DA, Sturm NR (2009) Hypermethylated cap 4 maxi- spliced leader cap 4 is mediated by a 48 kDa protein related to vaccinia virus VP39. mizes Trypanosoma brucei translation. Mol Microbiol 72(5):1100–1110. Mol Biochem Parasitol 147(1):137–139. 6. Parker R, Sheth U (2007) P bodies and the control of mRNA translation and degra- 25. Arhin GK, Li H, Ullu E, Tschudi C (2006) A protein related to the vaccinia virus cap- dation. Mol Cell 25(5):635–646. specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma 7. Dunckley T, Parker R (1999) The DCP2 protein is required for mRNA decapping in brucei. RNA 12(1):53–62. Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J 18(19): 26. Hall MP, Ho CK (2006) Functional characterization of a 48 kDa Trypanosoma brucei 5411–5422. cap 2 RNA methyltransferase. Nucleic Acids Res 34(19):5594–5602. 8. Wang Z, Jiao X, Carr-Schmid A, Kiledjian M (2002) The hDcp2 protein is a mammalian 27. Zamudio JR, et al. (2006) Complete cap 4 formation is not required for viability in mRNA decapping enzyme. Proc Natl Acad Sci USA 99(20):12663–12668. Trypanosoma brucei. Eukaryot Cell 5(6):905–915. 9. Jiao X, Chang JH, Kilic T, Tong L, Kiledjian M (2013) A mammalian pre-mRNA 5′ end 28. Silva E, Ullu E, Kobayashi R, Tschudi C (1998) Trypanosome capping enzymes display a capping quality control mechanism and an unexpected link of capping to pre-mRNA novel two-domain structure. Mol Cell Biol 18(8):4612–4619. processing. Mol Cell 50(1):104–115. 29. Hall MP, Ho CK (2006) Characterization of a Trypanosoma brucei RNA cap (guanine 10. Lykke-Andersen J (2002) Identification of a human decapping complex associated N-7) methyltransferase. RNA 12(3):488–497. with hUpf proteins in nonsense-mediated decay. Mol Cell Biol 22(23):8114–8121. 30. Banerjee H, et al. (2009) Identification of the HIT-45 protein from Trypanosoma brucei 11. Cao D, Parker R (2003) Computational modeling and experimental analysis of non- as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the sense-mediated decay in yeast. Cell 113(4):533–545. recombinant and endogenous proteins. RNA 15:1554–1564. 12. Muhlrad D, Parker R (1994) Premature translational termination triggers mRNA de- 31. Mildvan AS, et al. (2005) Structures and mechanisms of Nudix hydrolases. Arch Bio- capping. Nature 370(6490):578–581. chem Biophys 433(1):129–143. 13. Eulalio A, Behm-Ansmant I, Izaurralde E (2007) P bodies: At the crossroads of post- 32. Weng J, et al. (2008) Guide RNA-binding complex from mitochondria of trypanoso- transcriptional pathways. Nat Rev Mol Cell Biol 8(1):9–22. matids. Mol Cell 32(2):198–209. 14. Mukherjee C, et al. (2012) Identification of cytoplasmic capping targets reveals a role 33. Güther MLS, Urbaniak MD, Tavendale A, Prescott A, Ferguson MAJ (2014) High- for cap homeostasis in translation and mRNA stability. Cell Reports 2(3):674–684. confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope- 15. Schoenberg DR, Maquat LE (2009) Re-capping the message. Trends Biochem Sci 34(9): tag organelle enrichment and SILAC proteomics. J Proteome Res 13(6):2796–2806. 435–442. 34. Wang LK, Shuman S (2002) Mutational analysis defines the 5′-kinase and 3′-phos- 16. Schibler U, Perry RP (1976) Characterization of the 5′ termini of hn RNA in mouse L phatase active sites of T4 polynucleotide kinase. Nucleic Acids Res 30(4):1073–1080. cells: Implications for processing and cap formation. Cell 9(1):121–130. 35. Weitzer S, Martinez J (2007) The human RNA kinase hClp1 is active on 3′ transfer RNA 17. Spencer E, Loring D, Hurwitz J, Monroy G (1978) Enzymatic conversion of 5′-phos- exons and short interfering RNAs. Nature 447(7141):222–226. phate-terminated RNA to 5′-di- and triphosphate-terminated RNA. Proc Natl Acad Sci 36. Cohen LS, et al. (2005) Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is USA 75(10):4793–4797. sequence and context dependent. Mol Cell Biol 25(20):8779–8791. 18. Otsuka Y, Kedersha NL, Schoenberg DR (2009) Identification of a cytoplasmic complex 37. Milone J, Wilusz J, Bellofatto V (2002) Identification of mRNA decapping activities that adds a cap onto 5′-monophosphate RNA. Mol Cell Biol 29(8):2155–2167. and an ARE-regulated 3′ to 5′ exonuclease activity in trypanosome extracts. Nucleic 19. Agabian N (1990) Trans splicing of nuclear pre-mRNAs. Cell 61(7):1157–1160. Acids Res 30(18):4040–4050.

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