Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

ABSTRACT

Modified nucleosides in tRNA anticodon loops such as 5-methoxy-carbonyl-methyl-2-thiouridine (mcm⁵s²U) and pseuduridine (Ψ) are thought to be required for an efficient decoding process. In Saccharomyces cerevisiae, the simultaneous presence of mcm⁵s²U and Ψ38 in tRNA^Gln_UUG was shown to mediate efficient synthesis of the Q/N rich [PIN⁺] prion forming protein Rnq1.¹ In the absence of these two tRNA modifications, higher than normal levels of hypomodified tRNA^Gln_UUG, but not its isoacceptor tRNA^Gln_CUG can restore Rnq1 synthesis. Moroever, tRNA overexpression rescues pleiotropic phenotypes that associate with loss of mcm⁵s²U and Ψ38 formation. Notably, combined absence of different tRNA modifications are shown to induce the formation of protein aggregates which likely mediate severe cytological abnormalities, including cytokinesis and nuclear segregation defects. In support of this, overexpression of the aggregating polyQ protein Htt103Q, but not its non-aggregating variant Htt25Q phenocopies these cytological abnormalities, most pronouncedly in deg1 single mutants lacking Ψ38 alone. It is concluded that slow decoding of particular codons induces defects in protein homeostasis that interfere with key steps in cytokinesis and nuclear segregation.

Introduction

tRNA molecules are known to carry a variety of chemically distinct modified nucleosides that influence different aspects of tRNA function.² Modified bases present in the anticodon loop are in a prime position to modulate the efficiency of translation. In several cases where functionally non-redundant isoacceptor tRNAs are in place, only one of them naturally carries a particular modification. For example, 5-methoxy-carbonyl-methyl-2-thiouridine (mcm⁵s²U) is present at the wobble position (U34) in tRNAs reading the A-ending codons for Gln (CAA), Glu (GAA) and Lys (AAA), but not in the isoacceptors naturally reading the G-ending codons for the same aminoacids (CAG, GAG, AAG).³ Hence, the presence or absence of mcm⁵s²U could affect mRNA translation differentially depending on the frequency and relative use of the alternative A-ending/G-ending codons.⁴

The formation of mcm⁵s²U requires two genetically independent pathways, one depending on the Elongator complex and its interactors, the other depending on the sulfur transfer protein Urm1 and its sulfur donors (Nfs1, Tum1, Uba4) and acceptors (Ncs2, Ncs6).^5-8 Absence of either alone removes the mcm⁵- (Elongator) or the s²U part (Urm1) and induces overlapping defects including increased ribosomal frameshifting during mRNA translation, while elimination of both modification pathways severely aggravates these defects or may even cause synthetic lethality.^1,9-13 Further, complete absence of mcm⁵s²U was shown to cause a substantial ribosomal slow down at the A-ending codons for Lys and Gln.¹⁴ This triggers widespread aggregation of cellular proteins, affecting mostly components of larger protein complexes. The cellular proteins enriched in the aggregates of an mcm⁵s²U defective mutant are broadly overlapping with those identified in mutants lacking the ribosome associated chaperones Ssb1 and Ssb2 that facilitate co-translational protein folding.¹⁴ It was concluded that slow translation of A-ending Lys (AAA) and Gln (CAA) codons in the U34 modification-minus mutants induces aggregation of nascent polypeptides which in turn may destabilize large metastable protein complexes.¹⁴

Deg1 introduces a distinct modification, pseudouridine (Ψ), in various tRNA species at position 38 or 39, being part of the anticodon loop (Ψ38) or the loop-closing base pair (A31:Ψ39), respectively.¹⁵ In yeast, tRNA^Gln_UUG is the only tRNA species that carries mcm⁵s²U and also harbours a Deg1-dependent Ψ38, and negative genetic interaction data identified a strong growth defect in cells with defects in U34 modification and Ψ38/39 formation.^1,16,17 These phenotypic consequences are likely caused by a functional defect of tRNA^Gln_UUG upon removal of either of the two mcm⁵/s²U parts in combination with absence of Ψ38.^1,17

The yeast prion protein Rnq1 [PIN⁺] requires mcm⁵s²U and Ψ38 for efficient synthesis

The idea of specific functional impairment of tRNA^Gln_UUG in the combined absence of mcm⁵/s²U and Ψ38 was tested by analyzing translation of Rnq1, the protein forming the [PIN⁺] prion in the commonly used S288C derived BY4741 strains.¹⁸ The Rnq1 protein exhibits an extremely high Gln content (19% Gln) and since the majority of these residues (69%) are encoded by the CAA codon, Rnq1 synthesis was expected to depend strongly on the presence of mcm⁵s²U and Ψ38 in tRNA^Gln_UUG. Efficiency of Rnq1 synthesis was analyzed using a construct containing a GFP tagged version of RNQ1 encoding a truncated Rnq1^1-375 form containing the Q-rich prion domain of Rnq1 that was shown to form aggregation foci in [PIN⁺] cells and diffuse localization in [pin⁻] cells.¹⁹ In the context of tRNA modification mutants, however, so far only the translational efficiency of the RNQ1^1-375-GFP transcript was analyzed. Indeed, while loss of either mcm⁵/s²U or Ψ38 alone induced mild reduction in Rnq1^1-375-GFP protein biosynthesis, a severe defect was observed when both modifications were absent.¹ Thus, the Q-rich prion domain of Rnq1 is inefficiently synthesized in the analyzed tRNA modification double mutants. Whether or not such reduced synthesis affects the prion behavior of Rnq1 is currently under investigation. Since higher than normal levels of tRNA^Gln_UUG but not its isoacceptor tRNA^Gln_CUG rescue this translational defect, it is assumed that combined loss of both modifications specifically impairs the capacity of tRNA^Gln_UUG to decode CAA codons and translate transcripts encoding Gln rich proteins (Fig. 1). It remains to be investigated whether such downregulation of Rnq1^1-375-GFP levels in tRNA modification mutants changes the aggregation propensity of the protein and in turn affects the propagation of the [PIN⁺] state or impairs the induction of the [PSI⁺] prion, which requires the [PIN⁺] state of Rnq1.²⁰

Figure 1.

Scheme illustrating effects of dual tRNA modification loss on protein synthesis and subsequent effects. (A) Fully modified tRNA^Gln_UUG containing Ψ38 and mcm⁵s²U mediates efficient translation of mRNA encoding Q-rich protein such as the [PIN⁺] prion Rnq1. (B) Hyopmodified tRNA^Gln_UUG from elp3 deg1 mutants lacking Ψ38 (containing U38 instead) and the mcm⁵ side chain of the mcm⁵s²U modification. Efficiency of translation of mRNA encoding Q-rich protein is severely reduced.¹ This has negative effects on protein homeostasis, induces aggregation of other proteins and causes (among others) defects in cell polarity and cytokinesis.

Overlapping phenotypes of combined tRNA modification mutants

When analyzing phenotypes of tRNA modification mutants it was noted that mutants lacking mcm⁵ + s²U, mcm⁵/s²U and ct⁶A (cyclic N⁶-threonly-carbamoyl-adenosine)²¹ or mcm⁵/s²U and Ψ38/39 display common cytological phenotypes. These are characterized by the appearance of cell clusters with elongated and mal-positioned buds and go hand in hand with impaired actin cable and patch formation as well as nuclear segregation defects (Fig. 2). The defects subsequently result in formation of cells with multiple nuclei and fragile walls undergoing spontaneous cell lysis.¹ Strikingly, however, the tRNA modification mutant cultures display a mixture of normal and deformed cells, which suggests that these phenotypic transitions occur at a certain point of a cell's replicative lifespan, possibly related to cell ageing. Indeed, when consecutive replicative cycles of phenotypically normal virgin mutant cells were followed by micro-dissection, most mutant cells carried out this phenotypic transition after a number of normal cell divisions.¹ Hence, aged tRNA modification mutant cells are much more likely to accumulate cytological abnormalities compared to virgin or young mother cells. Interestingly, similar cytological phenotypes occur in mutants with inappropriate anticodon loop modification of tRNA^Lys UUU or tRNA^Gln_UUG, suggesting that the defect originates from tRNA malfunction in general, rather than specific malfunction of tRNA^Gln_UUG.

Figure 2.

Hallmarks of the cytological phenotype observed in dual tRNA modification mutants. Shown are wild type (WT) and deg1 elp3 mutant cells, stained with rhodamine-conjugated phalloidine (RHD) and DAPI. Left: RHD fluorescence visualizing the actin cytoskeleton, right: DAPI fluorescence indicating numbers and position of nuclei.

Protein aggregation mediated phenotypes

Since the cytokinesis defect occurs in mutants predicted to impair distinct tRNA species, it appears likely that it is not generated by the translational repression of similar proteins. As loss of mcm⁵s²U was already shown to induce protein homeostasis defects by ribosomal slow down,¹⁴ it appeared likely that combined modification defects involving ct⁶A or Ψ38/39 might induce a similar effect. Indeed, both tcd1 elp3 (lacking mcm⁵U and ct⁶A) as well as deg1 elp3 mutants (lacking mcm⁵U and Ψ38/39) strongly trigger protein aggregate formation compared to either single mutant or the wild type. To further substantiate the assumption that protein aggregates might represent the cause of terminal failure in cytokinesis after consecutive replicative cycles, an aggregating polyQ protein (Htt103Q) was expressed in wild type and deg1 mutant cells.²² Following induction of aggregating Htt103Q with a polyQ repeat of 103, a clear phenotypic transition from normal to elongated morphology was observed.¹ This effect was strongly aggravated in cells already lacking Ψ38/39 due to the deg1 mutation. In both cases not only a cytokinesis defect but also a clear nuclear segregation defect was observed, strongly resembling the morphological phenotypes of dual tRNA modification mutants (Fig. 2). Both the wild type as well as the deg1 mutant display normal morphology after expression of the non-aggregating Htt variant with a polyQ length of 25. These observations support the conclusion that the induction of endogenous protein aggregates in double tRNA modification mutants might directly cause these cytological abnormalities, since an aggregating protein itself is capable of triggering similar effects. This assumption is also consistent with the sudden onset of cytological defects depending on the replicative age of the mother cell, since protein aggregates are known to be accumulated in aging mother cells.²³ Possibly, tRNA modification mutants such as deg1 elp3 accumulate protein aggregates during replicative ageing and once a certain threshold is passed, key processes of cell division become disturbed.

Future directions

It is now established that induction of protein aggregation is a common consequence in tRNA modification mutants that either lack two independent modifications at the same base (mcm⁵/s²U) or parts of this modification in combination with other anticodon loop modifications (ct⁶A37 or Ψ38). The finding that in one of such mutants (elp6 ncs2), aggregates are similar to those accumulating in cells lacking the ribosome associated Ssb chaperone and include many subunits of metastable large protein complexes¹⁴ suggests that these are amorphous- rather than amylogenic (prion-like) aggregates, as the latter typically form by a single protein. Interestingly, however, different defects of the ribosome associated chaperone complex (RAC) induced in zuo1, ssz1 and ssb1 ssb2 mutants all increased de novo generation of the [PSI⁺] prion.^24-26 This may suggest that interference with translation at the level of tRNA functionality might impact on the tendency to form spontaneous amyloid aggregates as well, since similarities in protein homeostasis defects with the RAC mutants exist. However, dual tRNA modification loss affecting tRNA^Gln_UUG was also shown to severely reduce synthesis of at least one Q/N-rich prion, which might impair propagation and possibly de-novo generation of Q/N rich prions. Details about the mechanism how ribosomal slow down during the translation of transcripts enriched in mcm⁵s²/Ψ dependent codons might destabilize metastable proteins, the mRNAs of which are not enriched in those codons remain to be elucidated. Possibly, slow decoding of transcripts enriched in these mcm⁵s²/Ψ dependent codons causes initial misfolding of the nascent polypeptide that sequesters regulators of protein homeostasis which are most critical to maintain solubility of metastable protein complexes.¹⁴ Analyzing solubility changes of Rnq1 and rates of de-novo induction of [PIN⁺] in tRNA modification mutants with specific decoding defects of CAA (Gln) codons could possibly inform about the validity of these assumptions.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

FUNDING

We gratefully acknowledge funding support by DFG Priority Program SPP1784 ‘Chemical Biology of Native Nucleic Acid Modifications’ to RS (SCHA750/20) and RK (KL2937/1).