Rnase a

dsDNA, dsRNA and RNA:DNA hybrids were generated using 35 nt long 5'-Cy5 (for DNA) and 5'-Cy3 (for RNA) labeled oligonucleotides (Sigma) as described above (see RNA binding assays). Single-stranded and double-stranded nucleic acids were incubated with 10 U of RNaseH (Epicenter) in a buffer containing Tris-HCl pH 7.4 and 100 mM NaCl for 30 min at 37°C. RNaseA treatment was carried out with 10 μg of RNaseA (Fermentas) in a buffer containing Tris-HCl pH 7.4 and 350 mM NaCl (high salt) or 100 mM NaCl (low salt) for 30 min at 37°C. Reactions were stopped in a final concentration of 12 nM EDTA, 0.6% SDS and 48% glycerol and incubated on ice for 10 min. Oligonucleotides were diluted to a concentration of 200 nM before separation on a 10% native PAGE gel. Gels were pre-run for 30 min at 150 V at 4°C in 0.5X TBE and the samples were loaded in an equal volume of native loading buffer (30% (v/v) glycerol, 80 mM HEPES-KOH pH 7.9, 100 mM KCl, 2 mM magnesium acetate) and electrophoresed in 0.5X TBE at 150 V using an XCell Sure Lock Midi-Cell Electrophoresis System. After electrophoresis, the gels were scanned in a phosphorimager (GE Healthcare, Typhoon FLA 9500). Sequence information for the 5'- Cy5-DNA and 5'-Cy3-RNA MSR oligonucleotides are listed in Supplementary file 1 (RNaseA/H activity assay).

We performed the axon cultures as described above for RNA-seq analysis. Then, we treated the eyes with lysine- and arginine-free L15 (60%) medium for 1hr. After eye removal to eliminate the cell bodies, the axons were cultured in L15 depletion medium containing “heavy” amino acids (84 μg/ml [13C6,15N4] l-arginine, 146 μg/ml [13C6,15N2] l-lysine (Silantes, Germany) and Netrin-1 (600 ng/ml; R&D systems) for 3hrs. Soma removal was confirmed by absence of nuclear DAPI staining. For the preparation of control eye samples, we cultured dissected eyes in L15 depletion medium containing “heavy” amino acids at room temperature for 48hrs. Lysis of axons was performed using 500 μL Lysis buffer (9mM Tris-HCl pH 7.4, 270mM KCl, 9 mM MgCl₂, 1% n-octylglycoside (Sigma-Aldrich),100μg/ml cycloheximide (Sigma-Aldrich), 0.5mM DTT, EDTA-free protease inhibitor cocktail (Roche) and SUPERase In RNase Inhibitor (Ambion)). Lysates were centrifuged at 16.000 g at 4°C for 15 min and the supernatant was transferred to an ice-cold 1.5ml tube. For the puromycin/RNaseA/T1 treated sample, axons were treated with 200 μM puromycin for 15min before lysis and lysates were treated with 10 μg/μl RNase A (Ambion) and 250U RNase T1 (Ambion) for 15 min at 25°C.

The Thrdx-HA-mCherry and Thrdx-HA-insert-mCherry reporters were expressed in the PURExpress in vitro translation system (NEB, Ipswitch, MA) from PCR products. The peptidyl-tRNA construct was generated by creating a truncated mRNA lacking a stop codon directly after the Thrdx-HA sequence. The PURExpress reactions were initiated by mixing 1 μl of PCR product (29–22 ng/μl), 2 μl of solution A, 1.5 μl of solution B, and 0.6 μl of ³⁵S-methionine. The reactions were run for 45–60 min at 37°C. Following translation, the products were immediately heat-denatured and loaded on a 4–12% Bis-Tris gel at 4°C in XT-MES buffer. For the experiments in which the PURExpress reaction products were treated with RNase A (Figures 4B), 0.5–1 μg of RNase A (Ambion, Grand Island, NY) was added to each reaction and solutions were incubated on ice for an additional 30 min before being denatured and loaded on a gel. The peptide products of the PURExpress reactions were visualized by Phosphoimager and quantified with ImageQuant (Figure 3—figure supplement 2, and Figure 5—figure supplement 1).

Exponentially growing promastigote cells were washed with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄) and fixed in 1% (v/v) formaldehyde in 1X PBS for 5 min at room temperature. Cells were then treated with 0.1% Triton-X 100 in 1X PBS for 10 min and free aldehyde molecules were neutralized with 0.1 M glycine in 1X PBS for 10 min at room temperature. Cells that were treated with RNase A (Invitrogen) were washed with 1X PBS and then incubated with 20 µg of RNase A at 37°C for 30 min. Cells not treated with RNase A were washed with 1X PBS and incubated with 1X PBS at 37°C for 30 min. RNase A-treated and -non-treated cells were washed with 1X PBS and incubated with rabbit anti-LaTERT serum, obtained from recombinant Leishmania amazonensis TERT N-terminal region containing a putative telomerase RNA binding domain (TRD) (Giardini & Cano, unpublished data); LaTERT and LmTERT share about 95% identity (LmTERT) [12] (link)). α-LaTERT was diluted (1∶2000) in blocking solution (4% (w/v) bovine serum albumin) for 12 h at 4°C. Goat anti-rabbit IgG (2 mg/mL) labeled with Alexa Fluor 555 (Invitrogen) was diluted 1∶3000 and used as the secondary antibody. Cells were deposited on poly-L-lysine coated slides and used in fluorescence RNA in situ hybridization assays.