No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5'-OH ends phosphorylated by Trl1

Abstract

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3'-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5'-hydroxylated RNA fragments requiring 5'-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.

Fig. 1. Size characterization of 3′-NGD RNA fragments and ribosomal association.

a Schematic view of the URA3Rz mRNA showing the ribozyme (Rz) site (see also Supplementary Fig. 1a). Translational start (AUG) and stop codons are indicated. RNA1 (in magenta) is the stop-codon-less mRNA following ribozyme cleavage. Probes prA, prB and prC used in northern blots analysis are indicated. 5′ and 3′-NGD RNAs are the products of NGD cleavage of mRNA1. The lightning flash represents the NGD endonucleolytic cleavage and probe prA is designed for the detection of all potential 3’-NGD RNAs (see also Supplementary Fig. 1a). b Agarose gel electrophoresis followed by northern blot showing levels of mRNA1 and 3′-NGD RNA fragments in the indicated strains. The ScR1 RNA served as a loading control. c Analysis similar to b using 8% PAGE. The 5S rRNA served as a loading control. d Primer extension experiments using probe prA to determine the 5′-end of 3′-NGD RNAs. B1, B2, B3, B4 and B5 RNAs shown in c are indicated with the corresponding size in nucleotides (nts) calculated by primer extension. e Analysis of 3’-NGD RNA association with ribosomes in dom34 mutant cells. Upper, on ribosome profile, positions of 40S and 60S subunits, 80S and polysome are indicated. Panel below, 20 fractions were collected and extracted RNAs were analysed similarly to c. Bottom panel: 18S and 25S rRNAs are shown as references for ribosome and polysome sedimentation, using 1% agarose gel electrophoresis and ethidium bromide staining. f Distribution of the 3′-NGD RNAs analysed in e. For each fraction, levels of B1, B4 and B5 RNAs were plotted as a % of total amount of B1, B4 and B5 RNAs, respectively. The profile in e is reported in f. g Schematic view of the ribosome positioning on 3’-NGD RNAs combining information on size resolution, ribosomal association (Fig. 1), and RNase ribosomal protection of 3’-NGD RNAs (Supplementary Fig. 1). Codons are shown in black or magenta. A and P are ribosome A- and P-sites, respectively. Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.

Fig. 2. Dxo1 creates the heterogeneity of 3′-NGD RNA fragments in Xrn1-deficient cells.

8% PAGE followed by northern blot analysis using probe prA showing steady-state levels of RNAs in dom34 and other indicated mutant strains. The 5S rRNA served as a loading control. a Impact of DXO1 deletion on B2 and B3 RNA production. b Plasmid expression of wild-type Dxo1 (P^wt) or a Dxo1 catalytic mutant (P^mut) (mutant E260A/D262A). The vector control is plasmid pRS313 (Vec). Source data are provided as a Source Data file.

Fig. 3. Characterization of the endonucleolytic RNA fragments.

a Xrn1 digestion of total RNA extracts from dom34 mutant cells in the presence or absence of polynucleotide kinase (PNK) in vitro. 8% PAGE followed by northern blot analysis using probe prA. The 5S rRNA served as a loading control and 5.8S rRNA as a positive control of Xrn1 treatment. b Flow chart illustrating the method used for 3′-RACE as described in ref. with minor modifications according to McGlincy and Ingolia (see the Methods section). c PCR products obtained from 3′-RACE and separated on a 2% agarose gel. Purified DNAs for sequencing are indicated by an arrowhead. Prior to PCR, cDNAs were produced from total RNA from ski2, ski2/dom34 mutant cells expressing mRNA1. Control is made of total RNA from ski2/dom34 mutant cells without mRNA1 expression. d Sequences obtained after 3′-RACE performed on ski2 and ski2/dom34 total RNA. 100% of sequenced clones (omitting a residual 5S rRNA-linker amplification detected) have this DNA sequence. 5′-NGD DNA sequence (in green) and linker sequence (in magenta). Below, the site of mRNA1 is shown before and after the cleavage producing the 3’-NGD RNA B4 and the 3′-extremity of the 5′-NGD RNA confirmed by 3′-RACE. Source data are provided as a Source Data file.

Fig. 4. Analysis of the fate of 5′-NGD RNAs.

a Schematic model of mRNA1 before and after the endonucleolytic cleavages producing B4 RNAs. 5′-NGD resulting RNAs are shown here covered by two ribosomes and processed by Xrn1 to 48-nt RNAs (see also Supplementary Fig. 4 for 5′-NGD RNAs covered by three ribosomes). b 8% PAGE followed by northern blot analysis using probe prG showing steady-state levels of RNAs in the indicated mutant strains (left panel). Same membrane has been probed with prA as a ladder (right panel), and sizes of B5 (77nt), B4 (71 nt) and B1 (47nt) are indicated. The 5S rRNA served as a loading control. c Primer extension experiments using probe prG to determine the 5’-end of RNAs (see also Supplementary Fig. 5). d Schematic model of ribosome positioning on mRNA1 before and after the unique endonucleolytic cleavage producing B4 RNAs, localized 8 nts upstream of the first P-site nt. The position of disomes on the resulting 48-nt 5’-NGD RNA is shown with the distal ribosome having 1 nt in the A site (see also Supplementary Fig. 4). Source data are provided as a Source Data file.

Fig. 5. Endonucleolytically cleaved 5′-OH RNAs are phosphorylated by Trl1.

a 8% PAGE followed by northern blot analysis using probe prA. Levels of 3′-NGD RNA fragments in trl1/dom34 cells compared with those from TRL1/dom34 cells. b B1 and B4 RNA quantification relative to 5S rRNA from three independent experiments as shown in a. c 12% PAGE followed by northern blot analysis using probe prA. Treatment using T4 PNK to determine 5’-OH and 5’-P B4 RNA positions in the indicated strains. One-fourth of trl1/dom34 total RNA treated was loaded to limit scan saturation and allow TRL1/dom34 B4 RNA detection. The 5S rRNA served as a loading control. d As in Fig. 3a, Xrn1 digestion of total RNA extracts from trl1/dom34 mutant cells in the presence or absence of T4 PNK treatment in vitro. A minor band detected in trl1 is indicated by an asterisk (see also Supplementary Fig. 5 in which this band is detectable in TRL1 cells). Error bars indicate standard deviation (s.d.) calculated from three independent experiments. Source data are provided as a Source Data file.

Fig. 6. Identification of the endonucleolytic cuts on the NGD targeted (CGA)₄-mRNA.

a Schematic view of the (CGA)₄-mRNA. 5′- and 3′-NGD RNAs are products of NGD. The lightning flash represents the potential endonucleolytic cleavage upstream of the ribosome stall site. Probes prB and prH are indicated. 5′-NDG RNAs are shown processed by Xrn1 as described in Figs. 4a, d and Supplementary Fig. 4. b Northern blot analysis using probe prH showing steady-state levels of RNAs in the indicated mutant strains. Same membrane has been probed with prA as a ladder, and sizes of mRNA1 products, such as B5 (77 nt), B4 (71 nt) and B1 (47 nt) are indicated. Only the dom34 lane is shown. See Supplementary Fig. 6a for the sequence probed by prH. The 5S rRNA served as a loading control. Total RNA from WT cells without (CGA)₄-mRNA expression served as a control, noted C. A non-specific band is indicated by an asterisk. c Xrn1 treatment in vitro of total RNA from dom34 or dom34/ski2 mutant cells and northern blot using probe prH. The 5.8S rRNA is a positive control of Xrn1 treatment. d PCR products obtained from 3′-RACE (see also Fig. 3c). Prior to PCR, cDNAs were produced from cells expressing (CGA)₄-mRNA. Total RNA from cells without (CGA)₄-mRNA expression served as a control. e Sequences obtained after 3′-RACE performed in d on ski2/DOM34 total RNA. Sequence distribution is given in percentage. f Primer extension experiments using probe prJ to determine the 5′-end of RNAs. Xrn1-specific arrests are indicated by arrowheads. g Positioning of 3′-ends detected by 3′-RACE on (CGA)₄-mRNA from ski2/DOM34 cells (magenta arrowhead). Arrowhead sizes are proportional to the relative number of sequences obtained. Three cleavage clusters, C1, C2 and C3 were defined (see Supplementary Fig. 6d). Xrn1 arrests deduced from f are indicated by black arrowhead with sizes proportional to the intensity of reverse stops observed in f. h Schematic view of the ribosome positioning on (CGA)₄-mRNA deduced from Xrn1 arrests combined with the positioning of endonucleolytic cleavages provided by 3′-RACE. Source data are provided as a Source Data file.

Fig. 7. Model of No-Go decay pathway involving Trl1 kinase and 5’–3’ exoribonucleases.

Top of figure, the third ribosome is represented as competent for NGD endonuclease activation. We propose that the two first stalled ribosomes are not properly conformed to trigger the endonucleolytic process. NGD endonuclease cleavage (lightning flash) occurs 8 nts upstream of the first P-site residue, within the mRNA exit tunnel of the ribosome. Upstream ribosomes covering the resulting 5′-NGD fragments can advance and stall on the new 3′-end with 1 nt in the ribosomal A-site. Colliding ribosomes on this new RNA fragment can induce a novel NGD endonuclease activation. On 3′-NGD RNAs, like B4 RNAs, the NGD-competent ribosome dissociates and facilitates access of Trl1 RNA kinase to the 5′-hydroxylated 3′-NGD RNA, but we cannot exclude that the leading ribosome dissociates and upstream ribosomes run to form a new disome with 5′-protruding RNA. Once the RNA is 5′-phosphorylated, the processive 5′–3′ exonucleolytic activity of Xrn1 can degrade and produce B1 RNA. Alternatively, upon Xrn1 inactivation, 5′–3′ exonucleolytic digestion of this RNA by Dxo1 can occur and produce trimmed RNAs, such as B3 and B2 RNAs. Middle of figure, upstream of the 3rd ribosome, ribosomes are also competent for NGD endonuclease activation. Here, the endonucleolytic cleavage occurs in the 4th ribosome and B5 RNAs can derive from such RNAs after phosphorylation and 5′–3′ digestion. Bottom of figure, alternative pathways are proposed: B1 and B5 RNA production could be initiated by decapping or via Hel2-independent uncharacterized endo/exonucleolytic attacks.