A novel assay provides insight into tRNAPhe retrograde nuclear import and re-export in S. cerevisiae

Abstract In eukaryotes, tRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm where they serve as essential adaptor molecules in translation. However, tRNAs can be returned to the nucleus by the evolutionarily conserved process called tRNA retrograde nuclear import, before relocalization back to the cytoplasm via a nuclear re-export step. Several important functions of these latter two trafficking events have been identified, yet the pathways are largely unknown. Therefore, we developed an assay in Saccharomyces cerevisiae to identify proteins mediating tRNA retrograde nuclear import and re-export using the unique wybutosine modification of mature tRNAPhe. Our hydrochloric acid/aniline assay revealed that the karyopherin Mtr10 mediates retrograde import of tRNAPhe, constitutively and in response to amino acid deprivation, whereas the Hsp70 protein Ssa2 mediates import specifically in the latter. Furthermore, tRNAPhe is re-exported by Crm1 and Mex67, but not by the canonical tRNA exporters Los1 or Msn5. These findings indicate that the re-export process occurs in a tRNA family-specific manner. Together, this assay provides insights into the pathways for tRNAPhe retrograde import and re-export and is a tool that can be used on a genome-wide level to identify additional gene products involved in these tRNA trafficking events.


Strains and Plasmids
The mtr2 strain was obtained from the temperature-sensitive mutant collection, kindly provided by Dr. P. Heiter (The University of British Columbia), as described (1). The multicopy pRS426 plasmid expressing C-terminally protein A-tagged Mex67 was made as described previously (1). The Los1-MORF plasmid was obtained from the yeast ORF collection (3).

HCl/aniline assay
To induce wybutosine base excision, 10 µg of small RNA were incubated with 20 µl of 0.12 M HCl in a final volume of 50 µl dH 2 O for 3 hrs at 37 C. Following incubation, RNA/HCl samples were neutralized with 11.38 µl of 5 mM KOH. Controls lacking HCl contained only RNA in dH 2 0. Next, 12 µl of HCl-treated, neutralized RNA or HCl-untreated RNA was incubated with an equal volume of 0.5 M aniline, pH 4.5 at 60 C for 20 min to induce chain scission. RNAs were precipitated at -80 C overnight by adding: 450 µl dH 2 0, 47.5 µl 3M sodium acetate pH 5.2, 1410 µl cold 100% ethanol, and 1 µl GlycoBlue Coprecipitant (Invitrogen). RNA was centrifuged at 15,000 x g for 20 min, washed in 1 ml 70% ethanol, and dissolved in 20 µl dH 2 O.

Northern probes
Mature tRNA Phe and the 5' cleaved halves of tRNA Phe were detected using a digoxigeninlabelled probe that hybridizes to 18 nts at the 5' end of the 5' exon. To detect the 3' cleaved halves, an 18 nt digoxigenin-labelled probe was used that hybridizes to the 3' exon of tRNA Phe , with the 3' end of the probe complementary to the 3 rd nt at the 5' end of the 3' exon. The sequences of all probes used in this manuscript are as follows: tRNA Phe 5'/3' exon probe: 5' CGAACACAGGACCTCCAGATCTTCAGTCTGGCGCTCTCCC 3' tRNA Phe 5' exon probe: 5' CAACTGAGCTAAGTCCGC 3' tRNA Phe 3' exon probe: 5' TGCGAACTCTGTGGATCGAACACAGGACCT 3' tRNA Leu CAA 3' exon probe: 5' CTCTTGCATCTTACGATAC 5S rRNA probe: 5' GCACCTGAGTTTCGCGTATGGT 3'

SUPPLEMENTAL TABLES
Supplemental Supplemental Figure 1. Validation of the HCl/aniline assay to detect proteins involved in retrograde nuclear import as well as re-exporters of tRNA Phe . A) Small RNAs isolated from wild-type (WT) or tyw3 cells were treated with HCl for 0-5 hrs and subsequently incubated with aniline. Northern blot analysis was performed using the probe described in Fig Mature tRNA Phe levels are measured relative to 5S rRNA levels and all data are normalized to WT, which was set to 1. ** p < 0.01 relative to WT.
Supplemental Figure 3. Neither Los1 nor Msn5 overexpression rescues the tRNA Phe re-export defect in mex67-5 cells. A) Wild-type (WT) or mex67-5 cells expressing either a multicopy vector (V) or the multicopy plasmid containing the functional MEX67 gene C-terminally tagged with Protein A (Mex67-ProtA) (1), the functional LOS1 gene C-terminally tagged with MORF (2), or the MSN5 gene (untagged), were grown at 23 C (left) or shifted to 37 C for 2 hrs (right) (n = 2). Small RNAs were isolated and treated with or without HCl and subsequently incubated with aniline. Northern blot analysis was performed using a probe that hybridizes to the 5' exon. M: mature tRNA Phe ; 5'Ex: 5' exon of tRNA Phe . 5S rRNA levels serve as a loading control. B) The relative levels of tRNA Phe lacking wybutosine (yW) were calculated by dividing the amount of mature tRNA Phe (M) in the HCl-treated sample by the total amount of tRNA Phe (mature and cleaved) in that lane. Data are normalized to WT for the same temperature and WT is set to 1.
Supplemental Figure 4. Temperature-sensitive mtr2 cells display no detectable defect in the re-export of tRNA Phe . Wild-type (WT), mtr2 and tyw1 cells (n = 2) were grown at 23C or shifted to 37C for the time indicated. Small RNAs were isolated and treated with or without HCl and subsequently incubated with aniline. Northern blot analysis was performed using the probe shown in Fig. 1B. 5S rRNA levels serve as a loading control.