Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

Abstract

Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm⁵) and 5-carbamoylmethyl (ncm⁵) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm⁵ and mcm⁵ side chains at U₃₄ and their influence on Elongator activity.

The Yeast Elongator Complex has been Implicated in Many Cellular Processes

The Elongator complex in S. cerevisiae was first described to consist of 3 proteins (Elp1, Elp2, and Elp3), which were found to be associated with the hyperphosphorylated elongating form of RNA polymerase II (Pol II).¹ Furthermore, when introducing an elp1Δ strain into new growth conditions, mRNAs encoding gene products required for growth adaptation showed slow induction, supporting a defect in Pol II transcription and a nuclear localization of the complex.¹ Additional investigations identified the Elp4, Elp5, and Elp6 as a sub complex of Elongator complex.^2-4 In vitro, the Elp3 subunit of the 6-subunit Elongator complex was able to transfer acetyl groups from acetyl-CoA to histones and an Elp3p with amino acid substitutions in the C-terminal acetyl-CoA binding domain (HAT) show reduced histone acetylation.^5,6 In vivo, inactivation of the ELP3 gene resulted in decreased H3 and H4 acetylation.⁷ From these data it was concluded that Elongator complex was important for transcription elongation of Pol II and therefore it was named Elongator complex.^1,5-7 However, the involvement of Elongator complex in histone acetylation and transcription elongation was questioned, as chromatin immuno-precipitation experiments failed to detect Elongator on transcribing open reading frames and Elp1 to Elp3 proteins are localized in the cytosol.⁸

A cytoplasmic role of Elongator complex was suggested from the finding that Elp1p interacted with Sec2p, a protein required for polarized transport of secretory vesicles to the bud tip in S. cerevisiae.⁹ Transport of secretory vesicles requires the guanine nucleotide exchange factor Sec2p for activation of the vesicle-associated GTPase Sec4p.^10,11 Sec2p associates with Elp1p and it was proposed that the Elongator complex is required for regulation of exocytosis by influencing localization of Sec2p.⁹

A different nuclear function described for the Elongator complex was in telomeric gene silencing and DNA repair as Elongator mutants display partial loss of telomeric gene silencing and increased sensitivity to DNA damage agents.¹² In addition to the HAT domain, the Elp3p subunit of the Elongator complex shares sequence homology to proteins from the Radical S-adenosylmethionine (SAM) superfamily, proteins harboring an iron-sulfur cluster that catalyze a variety of radical reactions using SAM.¹³ Point mutations resulting in amino acid substitutions in the Radical SAM or HAT domains of the ELP3 gene displayed defects in telomeric gene silencing and DNA repair suggesting a role of Elongator complex in these processes.¹² A role for the Elongator complex in DNA repair was supported by its interaction with proliferating cell nuclear antigen (PCNA), a protein involved in DNA replication and DNA repair.¹²

Another function of Elongator complex, linking it to modification of tRNA, was based on the characterization of the Schizosaccharomyces pombe sin3-193 mutant. A sin3-193 mutant shows reduced levels of the modified wobble (U₃₄) nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) (Fig. 1)^14,15 causing an antisuppressor phenotype, i. e. the ochre serine tRNA suppressor encoded by the sup3-18 gene will no longer suppress the ade7-413 ochre allele.¹⁶ A sin3-193 mutant displays slight increase in cell volume, length, and amount of dead cells. This observed increase was independent of the presence or absence of the ochre tRNA suppressor, indicating that the Sin3 protein is required for proper cell cycle regulation.^14,15 The sin3⁺ gene was identified as an uncharacterized open reading frame (ORF).¹⁷ A strain with a null allele of the sin3⁺ gene lacks the wobble uridine nucleosides mcm⁵s²U in ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}UUC}^{Glu}$ and 5-methoxycarbonylmethyluridine (mcm⁵U) (Fig. 1) in the sup3-18 encoded ochre suppressor tRNA^Ser.¹⁷ The sin3⁺ gene encodes a conserved protein with 77% identity on the amino acid level to the S. cerevisiae Elp3 protein. The ELP3 gene of Saccharomyces cerevisiae is essential for formation of mcm⁵ and ncm⁵ side chains in mcm⁵s²U, mcm⁵U, 5-carbamoylmethyluridine (ncm⁵U) and 5-carbamoylmethyl-2'-O-methyluridine (ncm⁵U_m) at U₃₄ in tRNA (Fig. 1).¹⁷ Inactivation of the other S. cerevisiae Elongator genes encoding Elp1-Elp2p and Elp4-Elp6p displayed identical phenotypes as the elp3 null mutant including the tRNA modification defect.^17,18 This observation suggested that the Elongator complex is required for synthesis of the first step, an intermediate likely to be 5-carboxymethyluridine (cm⁵U)^19-23, in formation of mcm⁵U, mcm⁵s²U, ncm⁵U and ncm⁵U_m at U₃₄ in tRNA (Fig. 1).¹⁷ Additional support for a role in tRNA modification was recently strengthened by the observation that the Elp3p homolog from the archaea Methanocaldococcus infernus in vitro modifies U₃₄ in tRNA to cm⁵U in the presence of acetyl-CoA.²⁴

Figure 1.

Structure of 5-carboxymethyluridine (cm⁵U), 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), 5-carbamoylmethyluridine (ncm⁵U) and 5-carbamoylmethyl-2'-O-methyluridine (ncm⁵U_m). Highlighted in red are uridine side groups cm⁵, mcm⁵, ncm⁵, s² and the 2'-O-methylation of the ribose.

In Yeast, The Phenotypes of Elongator Deficient Cells are Linked to tRNA Modification

As the Elongator complex in yeast was implicated in 4 different cellular processes, it was important to determine if the complex has 4 distinct functions or if it affects one key process that leads to multiple downstream effects. In elongation of Pol II transcription, Elp3p was suggested to act as a HAT transferring acetyl groups to histones and indeed in an elp3 mutant there is a defect primarily in histone H3 acetylation.^5,7 The Elp3 protein was also suggested to be crucial for telomeric gene silencing and DNA repair.¹² In exocytosis, an interaction between Elp1p and Sec2p was proposed to be important for correct polarized localization of Sec2p.⁹ Interestingly, overexpression of various combinations of ${\text{tRNA}}_{{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$, ${\text{tRNA}}_{{\text{s}}^2\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{s}}^2\text{UUC}}^{\text{Glu}}$ restored acetylation of histone H3, telomeric gene silencing and DNA repair in an elp3 mutant and localization of Sec2p in an elp1 mutant.^18,25 In an elp3 mutant, the expression level of the Sir4 protein is decreased at translational level, a phenotype that is corrected if ${\text{tRNA}}_{{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$, ${\text{tRNA}}_{{\text{s}}^2\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{s}}^2\text{UUC}}^{\text{Glu}}$ are overexpressed.²⁵ The Sir4 protein is involved in assembly of silent chromatin at telomeres and in this gene AAA codons decoded by ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$ are overrepresented.²⁵ Based on these observations function of the S. cerevisiae Elongator complex is linked to tRNA modification/ translation and not transcription, exocytosis, telomeric gene silencing and DNA repair. The notion that mcm⁵s²U is important in ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$, ${\text{tRNA}}_{{\text{mcm}}^5{s}^2\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{mcm}}^5{\text{s}}^2\text{UUC}}^{\text{Glu}}$ was further supported by the observation that an ncs2 mutant unable to form the s² but not the mcm⁵ group shows identical but weaker phenotypes than Elongator deficient yeast cells. In S.cerevisieae, the Ncs2p in complex with Ncs6p is required for the final step in formation of the 2-thio group at wobble position in tRNAs ^26-28. Phenotypes observed in the ncs2 mutant are suppressed by overexpression of various combinations of ${\text{tRNA}}_{{\text{mcm}}^5\text{UUU}}^{\text{Lys}}$, ${\text{tRNA}}_{{\text{mcm}}^5\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{mcm}}^5\text{UUC}}^{\text{Glu}}$ (Table 1).^18,25 An explanation for the dependence of the mcm⁵s²U₃₄ nucleoside is that it promotes a canonical anticodon loop conformation which stabilize codon-anticodon interaction.^29,30

Table 1.

Phenotypes of S. cerevisiae and S. pombe mutants lacking wobble uridine modifications.

A. S. cerevisiae
Growth Defects	Deletion Mutants	Suppressed by tRNA*
Slow growth at 30°C	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Ts at 38°C	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Prolonged G1 phase	elp3Δ	tKUUU, tQUUG18
Adaptation to carbon source	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Sodium-chloride sensitivity	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Rapamycin Sensitivity	nsc2Δ, ncs6Δ, urm1Δ or uba4Δ	tKUUU, tQUUG, tEUUC28
Caffeine sensitivity	elp3Δ or ncs2Δ	tKUUU, tQUUG18
	nsc2Δ, ncs6Δ, urm1Δ or uba4Δ	tKUUU, tQUUG, tEUUC28
Diamide Sensitivity	nsc2Δ, ncs6Δ or urm1Δ	tKUUU, tQUUG, tEUUC28
	uba4Δ	tKUUU, tQUUG28
Transcription and Chromatin-Remodelling Defects	Deletion Mutants	Suppressed by tRNA*
GAL1 mRNA induction	elp3Δ or ncs2Δ	tKUUU, tQUUG18
ENA1 mRNA induction	elp3Δ	tKUUU, tQUUG18
Lethal in combination with histone H4 (4-19Δ)	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Ts in combination with histone H3 (3-29Δ)	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Ts in combination with histone H3 (K14R) / histone H4 (K8, 16R)	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Synergistic growth defect in combination with gcn5Δ	elp3Δ or ncs2Δ	tKUUU, tQUUG18
Acetylation defect of lys14 in histone H3	elp3Δ	tKUUU, tQUUG18
Secretion Defects	Deletion Mutants	Suppressed by tRNA*
Viable at 34°C in combination with sec2-59	elp1Δ or ncs2Δ	tKUUU, tQUUG18
Mislocalization of Sec2p	elp1Δ or ncs2Δ	tKUUU, tQUUG18
DNA Repair and Telomere Gene Silencing Defects	Deletion Mutants	Suppressed by tRNA*
Hydroxyurea (HU) sensitivity	elp3Δ or tuc2Δ	tKUUU, tQUUG, tEUUC25
Telomere gene silencing	elp3Δ or tuc2Δ	tKUUU, tQUUG, tEUUC25
tRNA Modification Defects	Deletion Mutants	Suppressed by tRNA*
mcm5, ncm5 or s2 modification defect	elp3Δ or ncs2Δ	No 18. 25
B. S. pombe
Growth Defects	Deletion Mutants	Suppressed by tRNA**
Ts at 36°C	ctu1Δ	tKUUU, tEUUC31
	elp1Δ, elp3/sin3Δ, elp4Δ, elp6Δ or ctu1Δ elp3/sin3Δ	tKUUU32
Rapamycin Sensitivity	elp1Δ, elp3/sin3Δ, elp4Δ, elp6Δ or ctu1Δ elp3/sin3Δ	tKUUU32
SDS Sensitivity	ctu1Δ elp3/sin3Δ	tKUUU32
H2O2 Sensitivity	elp3/sin3Δ or ctu2Δ	tKUUU33

In S. cerevisiae, tRNA tK_UUU(Lys), tQ_UUG(Gln) and tE_UUC(Glu) have mcm⁵s²U at wobble position.

In S. pombe, tE_UUC(Glu) has mcm⁵s²U at wobble position; identity of U₃₄ in tRNA tK_UUU(Lys) is unknown.

^* In S. cerevisiae, null mutations in Elongator genes (ELP1-ELP6) abolish formation of mcm⁵ side chain; null mutations in the NCS2/TUC2, NCS6, URM1 or UBA4 genes abolish formation of the s² moiety.

^** In S. pombe, null mutations in Elongator genes (elp1⁺, elp3⁺, elp4⁺ or elp6⁺) abolish formation of mcm⁵ side chain and the s² moiety (see text); null mutations in the ctu1⁺ or ctu2⁺ genes abolish formation of the s² moiety.

Additional support that elevated levels of hypomodified tRNA correct phenotypes observed in mutants with defects in wobble uridine modifications came from experiments in Schizosaccharomyces pombe. In S. pombe, elevated levels of hypomodified ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$ or ${\text{tRNA}}_{{\text{mcm}}^5{\text{s}}^2\text{UUC}}^{\text{Glu}}$ or combinations thereof suppress phenotypes observed in the ctu1Δ and ctu2Δ single mutants lacking the s²-group, the elp3/sin3Δ single mutant or the elp3Δ ctu1Δ double mutant lacking both modifications (Table 1).^31-33 In S. pombe Ctu1p is the homolog to Ncs6p and Ctu2p is the homolog to Ncs2p ³¹.

Kluveromyces Lactis γ-toxin a Tool to Identify Genes Required for Wobble Uridine Modifications

Another phenotype of yeast Elongator mutants is resistance to K. lactis killer toxin.^34-37 Certain strains of the dairy yeast K. lactis contains “killer DNA," a plasmid pair (k1 and k2) encoding a 3-subunit anti-yeast toxin complex known as zymocin.^38-40 Upon secretion, the α- and β-subunits dock the zymocin to the cell wall of susceptible yeasts and facilitate transfer of the cytotoxic γ-subunit, which will arrest the cell before START in the G1 phase of the cell cycle.^37,40,41 Two types of S. cerevisiae mutants resistant to zymocin have been described.³⁷ Type I resistant mutants are defective in binding and uptake of zymocin, but sensitive to endogenous expression of the γ-toxin. Type II mutants were believed to be target site mutants as they are resistant to both exogenous zymocin and endogenous expression of γ-toxin.^37,40 The cellular target(s) of K. lactis γ-toxin was unsolved for more than 20 y and initially adenylate cyclase was mistakenly identified as the target of γ-toxin.^41,42 The γ-toxin turned out to be an endonuclease having ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUC}}^{\text{Glu}}$, ${\text{tRNA}}_{{\text{mcm}}^5{\text{s}}^2\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$ as substrates.⁴³ These tRNAs have the mcm⁵s²U modified nucleoside at wobble position and the endonuclease cleaves the tRNAs between U₃₄ and U₃₅.⁴³ Presence of the mcm⁵ side chain of mcm⁵s²U is crucial for these tRNAs to be substrates, explaining why Elongator mutants are resistant to zymocin or endogenously expressed γ-toxin.^43,44 Thus, the γ-toxin resistance phenotype as well as phenotypes of yeast Elongator mutants suppressed by overexpression of hypomodified tRNAs are explained by an inability to make mcm⁵ and ncm⁵ side chains at wobble uridines.^18,43

There has been a number of genetic screens to identify mutants resistant to zymocin, generating iki mutants (insensitive to killer toxin), kti mutants (killer toxin insensitive) and tot mutants (toxin target).^34-37 Strains with mutations in any of the Elongator subunit genes (ELP1-ELP6), killer toxin insensitive genes (KTI11-KTI14), the TRM9 gene, the SIT4 gene or both the SAP185 and SAP190 genes are type II mutants ^34-37,40,45, and these mutants are unable to form mcm⁵ and ncm⁵ side chains at wobble position (Table 2).^17,46 To identify additional mutants affecting formation of the mcm⁵ group, a yeast deletion collection containing 4800 strains, with different non-essential gene deletions, was screened for resistance to zymocin.⁴⁶ In addition to strains deleted for ELP, KTI, SIT4, and TRM9 genes, 5 strains (urm1Δ, uba4Δ, ncs2Δ, ncs6Δ, and tum1Δ) were identified (Table 2). These strains lacked the s² group in mcm⁵s²U (Fig. 1), illustrating the importance of both the mcm⁵ and s² groups for the action of γ-toxin.

Table 2.

Genes having mutated alleles causing a defect in wobble uridine modifications.

S. cerevisiae (Gene name/Alias)	S. pombe (Gene name/Alias)	C. elegans (Gene name/Alias)	A. thaliana (Gene name/Alias)	M. musculus (Gene name)	H. sapiens (Gene name)
ncm5/mcm5 side chains
ELP1/IKI3/KTI7/TOT117	-	elpc-181	AtELP1/ELO286	Ikbkap85	IKBKAP87
ELP2/KTI3/TOT217	-	.	-	-	-
ELP3/KTI8//TOT3/HPA117	elp3+/sin3+17	elpc-381	AtELP3/ELO386	-	-
ELP4/KTI9/TOT7/HAP117	-	.	-	-	-
ELP5/IKI1/TOT5/HAP217	-	.	-	-	-
ELP6/KTI4/ TOT6/HAP317	-	-	-	-	-
KTI11/DPH3/KTI5 * 17, 46	-	dph-3103	-	-	-
KTI12/TOT417	-	-	-	-	-
KTI13/ATS1/FUN2817	-	-	-	-	-
KTI14/HRR2546	-	-	-	-	-
SIT4/PPH146	-	-	-	-	-
SAP185 ** 46	-	-	-	-	-
SAP190 ** 46	-	-	-	-	-
5-methoxy residue of mcm5U/mcm5s2U
TRM9/KTI120	-	-	AtTRM9 **** 53	Alkbh8 *** 22	-
TRM11221, 23	-	-	-	-	-
2-thio group (s2) of mcm5s2U
NFS1/SPL1104	-	-	-	-	-
ISU1/NUA1105	-	-	-	-	-
ISU2/NUA2105	-	-	-	-	-
CFD1/DRE3105	-	-	-	-	-
NBP35 105	-	-	-	-	-
CIA1105	-	-	-	-	-
URM126-28, 46	-	-	-	-	-
UBA4/YHR126-28, 46	-	moc-3103	-	-	-
NCS6/YGL210W-A/TUC126-28,46, 106	ctu1+31	ctu-1/tuc-1/tut-128, 31, 81	-	-	-
NCS2/TUC218, 26-28, 46	ctu2+31	-	AtCTU2107	-	-
TUM127, 28, 46	-	-	-	-	-

^* KTI5 is a dominant allele of KTI11.

** A sap185Δ sap190Δ double mutant lacks ncm⁵/mcm⁵ side chains.

*** M. musculus Alkbh8 encodes the ALKBH8 protein containing a RNA recognition motif (RRM), AlkB-like dioxygenase domain (AlkB) and methyltransferase (MT) domain. MT domain catalyses methylation of cm⁵U into mcm⁵U together with TRM112; RRM/AlkB domains catalyse hydroxylation of mcm⁵U into (S)-mchm⁵U.

**** A. thaliana AtTRM9 encodes the AtTRM9 protein catalysing the methylation of cm⁵U into mcm⁵U together with AtTRM112a/b; AtALKBH8 catalyses hydroxylation of mcm⁵U into (S)-mchm⁵U.

Proteins Required for Formation of Wobble Uridine Modifications

Synthesis of the mcm⁵ side chain at the wobble position requires 15 gene products and formation of the s² group in mcm⁵s²U requires 11 gene products (Fig. 2). Strains with a deletion of any of the ELP1-ELP6, KTI11, KTI12, KTI14, SIT4 or SAP185 and SAP190 genes lack the mcm⁵U, mcm⁵s²U and ncm⁵U nucleosides, whereas a kti13 deletion mutant has severely reduced levels of these nucleosides.^17,46 No intermediates of mcm⁵U, and ncm⁵U are detected in any of the mutants, whereas s²U is detected in tRNAs normally containing mcm⁵s²U.^17,46 Thus, all these gene products are required for an early step in synthesis of mcm⁵ and ncm⁵ groups (Fig. 2).

Figure 2.

Proteins required for formation of the 5-methoxycarbonylmethyl (mcm⁵), 5-carbamoylmethyl (ncm⁵) and the 2-thio (s²) side groups on uridines at wobble position. Acetyl-CoA acts as a donor in formation of the cm⁵ side group. Trm9p and Trm112p utilize AdoMet (S-Adenosylmethionine) as a methyl donor to form the mcm⁵ side group. Last step in formation of ncm⁵ is unknown (?). R represents ribose and highlighted in red are uridine side groups cm⁵, mcm⁵, ncm⁵ and s². For details, see text.

The Elongator complex consists of 2 sub complexes, Elp1-Elp3p and Elp4-Elp6p ^1–4, and recently the crystal structure of the Elp4-Elp6p sub complex was solved at 2.1Å resolution.⁴⁷ The crystal structure revealed that all 3 subunits share a RecA-like fold and 2 heterotrimers form a hexameric ring like structure. A functional Elongator complex was proposed to contain 2 copies of the Elp1-Elp3p core complex on each hexameric Elp4-Elp6p ring.⁴⁷ Hexameric RecA-like ATPases of ring-translocases have the ability to bind specific DNA or RNA substrates.⁴⁸ Experiments supporting a direct role of Elongator complex in tRNA binding are; (i) a tRNA that should obtain a mcm⁵ group at wobble position co-precipitated with Elp1p or Elp3p¹⁷, (ii) in electrophoretic mobility shift assay (EMSA) experiments, the Elp4-Elp6p sub complex binds tRNA⁴⁷, (iii) the C-terminal domain (CTD) of Elp1 binds tRNA in an EMSA experiment.⁴⁹ The catalytic activity of Elongator complex is believed to reside in the Elp3 protein due to the presence of 2 domains. One sharing homology to acetyl-CoA binding (HAT) domains and the other to Radical S-adenosylmethionine (SAM) domains harboring an iron-sulfur (Fe-S) cluster that catalyze a variety of radical reactions by using SAM.^5,13,50 The presence of a Fe-S cluster in the Radical SAM domain and ability to bind SAM has been verified for the archaeal M. jannaschii Elp3p homologue.⁵¹ Homologues to the Elp3 protein are found in essentially all archaea but not the other subunits of the Elongator complex. ^{24, 51} This suggests that archaea require only Elp3p for formation of cm⁵U. In vitro, recombinant Elp3p from Methanocaldococcus infernus catalyze the transfer of an acetyl radical forming cm⁵U in the presence of acetyl-CoA, the reducing agent sodium dithionite and S-adenosylmethionine.²⁴ Thus, this unique enzymatic reaction mechanism explains the requirement of the SAM and HAT domains in Elp3p.

The last step in formation of mcm⁵U requires the tRNA methyltransferase Trm9 and the Trm112 protein, whereas no gene product responsible for the last step in formation of ncm⁵U is known (Fig. 2).^20-23,52,53 Other than being a subunit required for Trm9p activity, Trm112p is also a subunit required for the activity of 3 other methyltransferases, the tRNA methyltransferase Trm11p forming m²G₁₀ in tRNA, the ribosomal methyl transferase Bud23p methylating G1575 in 18S rRNA⁵⁴ and Mtq2p methylating the release factor eRF1.^52,55

A set of proteins associated with Elongator complex are suggested to regulate its activity. These are the Kti11-Kti14, Sit4, Sap185 and Sap190 proteins (Fig. 3). The Sit4p is a type 2A protein phosphatase that associates with members of a protein family termed SAPs.^56,57 Deletion of all 4 SAP genes (SAP4, SAP155, SAP185 and SAP190) confers the same phenotypes as loss of SIT4.⁵⁶ SAPs fall into 2 groups based on their sequence similarity: the SAP4/ SAP155 group and the SAP185/ SAP190 group.⁵⁶ The 2 groups of SAPs are believed to be either effectors or activators of Sit4p.⁵⁶ Sit4p, Sap185p, and Sap190p have been shown to physically interact with Kti14p.⁵⁸ KTI14 (HRR25) encodes a homologue to the mammalian casein kinase 1δ (CK1δ).⁵⁹ Kti14p interacts with Elongator and the interaction is dependent on Kti12p.⁶⁰ The kinase Kti14p and the phosphatase Sit4p seem to antagonistically regulate activity of Elongator complex by phosphorylation/ de- phosphorylation of the largest Elongator subunit Elp1p.^60,61 In addition to phosphorylation/ de- phosphorylation, the activity of Elongator complex also seems to be regulated by proteolysis of Elp1p.⁶² The Kti11p interacts with Elongator complex through its C-termini and loss of Kti11p enhances the proteolysis of Elp1p.^62,63 Except for being crucial for ncm⁵ and mcm⁵ side chain formation, Kti11p/Dph3p is also required for biosynthesis of the posttranslational modification diphtamide, a unique target on translation elongation factor 2 (eEF2) for bacterial ADP-ribosylating toxins.⁶⁴ In diphthamide biosynthesis, Kti11p is an electron donor for the Fe-S clusters in the Dph1-Dph2p⁶⁵, and it is conceivable that in the tRNA modification reaction Kti11p also acts as an electron donor for the Fe-S cluster in Elp3p. The Kti11p also interacts with Kti13p a protein with structural features similar to the guanine exchange factors.⁶⁶ However, the role of Kti13p in yeast is not yet elucidated.

Figure 3.

Elongator and Elongator associated proteins. Elongator complex is suggested to contain 2 copies of the Elp1-Elp3p core complex (dark gray) on each hexameric Elp4-Elp6p ring.⁴⁷ Elongator associated proteins are clustered according to interaction studies. P indicates phosphorylation of Elp1p. For details, see text.

Formation of the 2-thio group present in ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUC}}^{\text{Glu}}$, ${\text{tRNA}}_{{\text{mcm}}^5{\text{s}}^2\text{UUG}}^{\text{G}\ln }$ and ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}{\text{s}}^{\text{2}}\text{UUU}}^{\text{Lys}}$, requires 11 gene products (Fig. 2) and the thiolation reaction has been successfully reconstituted in vitro.^26-28 In S. cerevisiae, presence of a mcm⁵ side chain at U₃₄ is a prerequisite for efficient 2-thio group formation and in an S. pombe elp3/sin3 mutant the 2-thio group formation is abolished.^26-28,32

Recently it was suggested that levels of certain modified nucleosides in tRNA, among them ncm⁵U, mcm⁵U and mcm⁵s²U could be altered in response to various stress conditions.⁶⁷ However, the levels of the ncm⁵U, mcm⁵U and mcm⁵s²U nucleosides were not quantified in individual tRNA isoacceptors rather they were quantified in bulk tRNA. Therefore, it is not possible to distinguish between regulation of modification on individual tRNA isoacceptors or if the levels of the tRNA isoacceptors are altered.

Role of Wobble Uridine Modifications in Translation

The wobble uridine nucleosides mcm⁵U and ncm⁵U were believed to either restrict pairing to A ⁶⁸ or to allow efficient interaction with both A and G.⁶⁸ Presence of the s² group in the wobble nucleoside mcm⁵s²U has been suggested to restrict reading to A-ending codons.^68,69 In S. cerevisiae, there are 42 different cytoplasmic tRNA species and 11 of these contain ncm⁵U, ncm⁵Um, mcm⁵U, or mcm⁵s²U at wobble position (Fig. 4).^70-73 Thus in yeast mutants affecting formation of ncm⁵ and mcm⁵ side chains, like Elongator mutants, these side chains are abolished in about 25% of the tRNA population which is a likely explanation for the pleiotropic phenotypes. As mutants defective in formation of mcm⁵/ncm⁵ or s² groups at wobble uridines are available, the in vivo roles of these modifications have been investigated using a set of different strategies.

Figure 4.

The genetic code and distribution of cytoplasmic S. cerevisiae tRNAs. The anticodon sequences of the 42 different tRNA species (1 initiator and 41 elongator tRNAs) are indicated.^43,69-72 For anticodons with an uncharacterized RNA sequence, the primary sequence is shown. The initiator and elongator tRNA^Met species have identical anticodon sequences. The wobble rules suggest that an inosine (I₃₄) residue allows paring with U, C, and sometimes A. A tRNA with a G or its 2′-O-methyl derivative (Gm) at the wobble position should read U- and C-ending codons. Presence of a C₃₄ residue or its 5-methyl (m⁵C) or 2′-O-methyl (Cm) variant should only allow pairing with G. The pseudouridine (Ψ)-containing tRNA^Ile is presumably unable to pair with the methionine AUG codon. The anticodons containing mcm⁵U, mcm⁵s²U, ncm⁵U and ncm⁵U_m derivatives are shown in bold. Copyright © American Society for Microbiology, [Molecular and Cellular Biology, 28, 2008, 3301–3312 and doi:10.1128/MCB.01542-07].⁷²

In one strategy, strains with specific tRNA gene deletions in combination with deletion of genes responsible for formation of mcm⁵/ ncm⁵ or s² groups were constructed and the growth properties of the strains were studied.⁷³ These analyses revealed that mcm⁵ and ncm⁵ side chains promote pairing with G-ending codons in most codon boxes and that concurrent mcm⁵ and s² groups improve reading of both A- and G- ending codons.⁷³

A second in vivo strategy is to quantify the role of wobble uridine modifications in a tRNA isoacceptor decoding the cognate A or the near cognate G ending codon using reporter systems. The SUP4-encoded ochre suppressor tRNA, where the primary anticodon sequence is UUA, has a mcm⁵ side chain at U₃₄.¹⁷ In an elp3 mutant lacking the entire mcm⁵ side chain resulting in an unmodified U₃₄, efficiency of decoding the cognate ochre UAA and the near cognate amber UAG codons by the SUP4 suppressor tRNA is reduced.⁷³ Also ${\text{tRNA}}_{{\text{mcm}}^{\text{5}}\text{UCU}}^{\text{Arg}}$ having the anticodon sequence UCU has a mcm⁵ side chain at U₃₄. In a trm9 mutant lacking the methyl esterification of cm⁵U₃₄, efficiency of decoding of multiple cognate AGA and multiple near cognate AGG codons by ${\text{tRNA}}_{{\text{cm}}^{\text{5}}\text{UCU}}^{\text{Arg}}$ is decreased.⁷⁴

The proteome expression in S. cerevisiae urm1 and uba4 mutants lacking the s² group of mcm⁵s²U or an elp3 mutant lacking ncm⁵ and mcm⁵ side chains has been investigated using stable isotope-labeling of amino acids in cell culture (SILAC) technology.⁷⁵ In urm1 and uba4 mutants about 270 proteins were either up- or down-regulated. The analysis revealed that 85% of up regulated proteins and 75% of down regulated proteins from the elp3 mutant overlap with the results from the urm1 and uba4 mutants. Codons AAA, CAA and GAA read by tRNAs having the mcm⁵s²U₃₄ modified nucleoside were enriched in the downregulated genes. Reporter constructs having any of these codons in multiple copies show reduced expression in urm1Δ, uba4Δ and elp3Δ backgrounds.⁷⁵ However, no correlation to mRNA levels of down regulated genes was reported.

Ribosome profiling (Ribo-seq) is a method to determine genome wide distribution of ribosomes at the codon level on mRNA.⁷⁶ Ribo-seq and RNA-seq (RNA Sequencing) analysis were done in S. cerevisiae strains lacking the mcm⁵ and ncm⁵ side chains (elp3 mutant), the s² group (ncs2, ncs6 and uba4 mutants), and a wild type strain.⁷⁷ Mutants with a defect in s² group formation show an accumulation of ribosomes when AAA and CAA codons were in the A-site, whereas the elp3 mutant lacking the mcm⁵ and ncm⁵ side chain, show ribosome accumulation at CAA and GAA codons. Although accumulation of ribosomes at these codons suggests a slowdown in translation, a correlation with a reduction in protein output could not be established.

Another tool for genome wide analysis in Schizosaccharomyces pombe is the fission yeast integrated ORFeome library, which is a library of 4910 open reading frames having a Flag2-His6 tag, where expression of constructs can be determined using anti-His-antibody.⁷⁸ A null allele of elp3⁺/ sin3⁺ gene was introduced into the ORFeome library and expression levels of the tagged ORF's were analyzed in elp3/ sin3Δ and wild type background.³² About 500 genes enriched in AAA and GAA codons showed reduced expression in elp3/ sin3Δ compared to wild type background. To investigate the relevance of AAA codons for expression, a candidate gene cdr2⁺ was chosen, where the AAA codons were mutagenized to AAG codons read by ${\displaystyle tRN{A}_{CUU}^{Lys}}$ having no Elongator dependent tRNA modification. In elp3/ sin3Δ background, expression of the protein encoded by the modified cdr2 gene improved significantly, suggesting that the expression was no longer Elongator dependent.³² With a similar strategy, in the S. pombe atf1⁺ gene AAA codons were changed to AAG codons and a similar result was obtained.³³

Elongator Complex in Multicellular Eukaryotes

Orthologues of the Elongator complex proteins can be found in multicellular eukaryotes and 6 subunit protein complexes have been purified from humans and plants.^3,79-85 Consistent with yeast, mutations in Elongator genes in the mouse M. musculus, the worm C. elegans, the plant A. thaliana and human cause defects in formation of wobble uridine modifications (Table 2).^82,86-88 In vitro, Elongator complex purified from HeLa cells have histone H3 and H4 acetyltransferase activity and depletion of the human homolog hELP1, IKAP (I kappa B kinase complex associated protein) in fibroblasts, leads to hypoacetylation of histone H3, transcription impairment and cell migration defects.^79,89,90 Mutations in genes encoding Elongator subunit proteins are associated to human diseases.⁹¹ Point mutations in the IKBKAP gene encoding IKAP cause familial dysautonomia (FD), a severe human hereditary neurodegenerative disorder.^92,93 Levels of the mcm⁵s²U nucleoside in tRNA isolated from brain tissue and fibroblast cell lines were 64–71% in FD patients compared to non-FD individuals.⁸⁸ As mice homozygous ikbkap^-/- knockouts are embryonic lethal, a complete loss of modified wobble uridine nucleosides in FD patients was not expected. ⁹⁴ Furthermore, a conditional inactivation of Ikbkap in mice leads to meiotic defects during spermatogenesis.⁸⁶ Also observed in mice is that knockdown of the Elp1p, the Elp3p or the Elp4p homologues in oocytes impairs zygotic paternal genome demethylation.⁸¹ In the fruit fly D. melanogaster, the Elongator complex has been implicated in several processes such as, larval- and neuro-development.^95,96 An explanation for the neurodevelopmental defects in D. melanogaster could be that Elongator complex acetylate Bruchpilot, a protein important for neuronal differentiation.⁹⁷ Both in mice and C. elegans, the Elp3p homolog has been implicated in acetylating lysine 40 (K40) in α-tubulin.^98,99 However, the C. elegans elpc-3 mutant did not have a defect in α-tubulin K40 acetylation.⁸² Later studies revealed MEC-17 and its paralogue ATAT-2 as the sole α-tubulin K40 acetyltransferases in C. elegans as single mec-17 and atat-2 mutants show reduced levels of α-tubulin K40 acetylation, whereas the double mec-17 and atat-2 mutants lack the K40 acetylation of α-tubulin.^100,101 The elpc-1, elpc-3 and tuc-1 mutants cause defects in translation in C. elegans.⁸² ELPC-1::GFP and ELPC-3::GFP reporters are strongly expressed in a subset of chemosensory neurons required for salt chemotaxis learning.⁸² The elpc-1 or elpc-3 gene inactivation causes a defect in this process, associated with a posttranscriptional reduction of neuropeptide and a decreased accumulation of acetylcholine in the synaptic cleft. The elpc-1 and elpc-3 mutations are synthetic lethal with the tuc1 mutation at 25°C and double mutants display developmental defects.⁸² In the plant Arabidopsis thaliana, mutations in the ELP1, ELP3, ELP4, or KTI12 gene homologues cause cell proliferation defects.^83,102

Conclusions and Perspectives

Independent of organism, Elongator mutants show very pleiotropic phenotypes. This has led to a debate whether Elongator complex has multiple functions or if its participation in one cellular process results in multiple downstream phenotypes. As Elp3 protein contains Radical SAM and HAT domains binding S-adenosylmethionine and acetyl-CoA, one theme is that Elongator complex is important for acetylation. Five substrates targeted for acetylation have been described, histones H3 and H4, α-tubulin, the neural differentiation protein Bruchpilot and lately a unique transfer of an acetyl radical to form cm⁵U₃₄ in tRNA. In yeast there is convincing evidence that the only function of the Elongator complex is in formation of the intermediate cm⁵U in biosynthesis of ncm⁵U and mcm⁵U side chains present at wobble position in 11 out of 42 tRNA isoacceptors.⁷³ This means that a substantial amount of the tRNA pool is missing wobble uridine modifications, implicating a defect in decoding during translation. All investigated phenotypes except the wobble uridine modification defect of Elongator mutants are efficiently suppressed by overexpressing tRNAs that in the wild type strain have the modified nucleoside mcm⁵s²U. This is an example of high copy suppression, i. e. when these tRNAs are overexpressed they compensate for the defect of the initial mutation. In this case higher levels of hypomodified tRNAs most likely compensate for the reduced codon/ anticodon interaction during translation due to lack of modifications at wobble position. Consistent with a Elongator tRNA dependent defect in translation, changing codons in a gene that are read by tRNAs containing modified wobble uridines to synonymous codons read by tRNAs not containing modified wobble uridines, restore protein expression, i.e. protein expression becomes Elongator independent.

In multicellular organisms, there has been no experiment where phenotypes observed in Elongator mutants have been suppressed by tRNA high copy suppression. However, as the role of Elongator in tRNA modification is conserved in eukaryotes it is likely that observed phenotypes are secondary to its primary function in tRNA modification.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Drs Marcus Johansson and Jan Larsson for comments on the manuscript.

Funding

A.S.B. is supported by grants from the Swedish Cancer Foundation (13 0301), Swedish Research Council (621-2012-3576) and Karin and Harald Silvanders Foundation (223-2808-12).