Figures and data
PARE identifies known Rnt1 cleavage sites and substrates.
(A) Schematic of PARE workflow. Total RNA is isolated from RNT1 and rnt1Δ strains. T4 RNA ligase ligates an adapter (red rectangle) onto exposed 5’ phosphates (red P’s) resulting from cleavage or decapping. Next-generation sequencing is performed from the 5’ adapter, resulting in reads that begin at the first nucleotide after the cleavage or decapping site. (B) >80,000 sites were detected with reads ≥1 cpm in the RNT1 strain (x-axis). 496 of these were decreased in the rnt1Δ strain (log2(FC)>4 blue, green, and yellow dots). Known ncRNA processing sites are shown in green, and known mRNA sites are shown in yellow. Shown are the averages of two independent biological replicates. (C) 496 putative Rnt1 cleavage sites cluster into 166 different substrates. All known Rnt1 ncRNA targets are detected as well as 2 novel snoRNA targets. 63 mRNA targets are detected, of which 3 are known and 60 are novel. Other sites detected include intergenic, antisense, intronic, and 5’ and 3’ UTR sites. (D) PARE detects known Rnt1 cleavage sites in ncRNA targets, validating PARE as a reliable method for identifying novel Rnt1 cleavage sites. Some known mRNA sites are also detected, but most have reads <1 cpm in RNT1. (E) PARE precisely detects the known Rnt1 cleavage site in pre-SNR83 (red arrowhead) located 61 nts upstream of its mature 5’ end (green arrowhead) on the 5’ side of an AGUU stem loop. PARE additionally reveals a novel site (pink arrowhead) located on the 3’ side of stem. Structure of the stem loop and IGV PARE screenshot are shown. Northern blot using a probe that hybridizes to mature SNR83 (grey bar) was performed in duplicate.
PARE identifies novel Rnt1 mRNA targets.
(A-D) PARE screenshots of Rnt1-cleaved mRNA targets. Strong peaks for Rnt1 cleavage (red arrowheads) are detected in (A) the known mRNA target BDF2 and novel targets (B) CAF4, (C) YDR514C, and (D) MTM1. PARE was performed in duplicate and both independent biological replicates are shown. (E) Northern blots detect cleavage products of BDF2 and CAF4. Shown is a representative of two independent biological replicates. PGK1 was used as a loading control. (F) Sequence alignment of 42 of 63 mRNA tetraloops and surrounding sequences.
Rnt1 directly and independently cleaves mRNAs.
(A) Validation of Rnt1 catalytic mutant by growth assay and by northern blot of SNR83. Growth assay strains were spotted on SC-Leu. Experiment was performed using two independent biological replicates. (B) Rnt1 catalytic mutant PARE. Wild-type RNT1 or rnt1-D245R cloned into a plasmid, or empty vector, was expressed in a rat1-ts xrn1Δ rnt1Δ triple mutant strain. PARE panels of BDF2, CAF4, and YDR514C are shown. Experiment was performed using three independent biological replicates. (C) Schematic of in vitro PARE workflow: RNA was isolated from a rnt1Δ-only strain and incubated with 0, 4, or 8 pmol of recombinant Rnt1. This RNA was then analyzed by PARE. (D) In vitro Rnt1 cleavage of BDF2, CAF4, and YDR514C (red arrowheads, Rnt1 cleavage sites detected in vivo; grey arrowheads, additional Rnt1 cleavage sites detected in vitro, but not in vivo). In vitro PARE was performed using two independent biological replicates. (E) Comparison of the numbers of in vivo and in vitro targets. (F) In vitro Rnt1 cleavage sites in MIG2 (panels 3-5) compared to in vivo Rnt1 cleavage sites in MIG2 (panels 1-2).
Localization is a key determinant in Rnt1 mRNA selection and cleavage.
(A) Confirmation of Rnt1 cytoplasmic relocalization by confocal fluorescence microscopy. RNT1-GFP or rnt1-ΔNLS-GFP cloned into a plasmid, or empty vector, was expressed in a rnt1Δ-only strain. The nucleus was stained with DAPI, pseudo-colored red. Experiment was performed using two independent biological replicates. (B) PARE of BDF2 and CAF4 cleaved by cytoplasmic Rnt1. Wild-type RNT1, rnt1-ΔNLS-GFP, or rnt1-K45I was expressed in a rat1-ts xrn1Δ rnt1Δ triple mutant strain. Experiments were performed using two independent biological replicates. (C) Northern blot of BDF2 and CAF4 cleaved by cytoplasmic Rnt1. PGK1 was used as a loading control.
Rnt1-cleaved mRNAs are subsequently degraded by Xrn1.
(A) Rnt1 cleavage peaks in BDF2, CAF4, and YDR514C in xrn1Δ-only PARE data. Two independent biological replicates are shown. PARE dataset from the rat1-ts xrn1Δ double mutant background is also shown for comparison. (B) Northern blot of BDF2 and CAF4 using RNA from wild-type RNT1, rnt1Δ, rat1-ts, and xrn1Δ single mutant strains. Experiment was performed using two independent biological replicates. PGK1 was used as a loading control.
Rnt1 and decapping products are derived from mRNAs with distinct poly(A) status.
IGV screenshots of RNT1 vs rnt1Δ PARE data generated from poly(A)-enriched and poly(A)-depleted samples show different distributions for Rnt1 products (red arrowheads) and decapping products (grey bars). The BDF2 mRNA is also a substrate for spliceosome-mediated decay (SMD, purple arrowhead).
Rnt1 cleavage of YDR514C mRNA contributes to normal cell growth.
(A) Schematic of rnt1Δ experimental evolution. Thirteen cultures of rnt1Δ were grown to saturation, then sub-cultured to a 1:1000 dilution for 10 cycles. Solid media growth assays were performed, and strains showing enhanced growth compared to the rnt1Δ parent strain were analyzed by whole-genome sequencing (WGS). The growth assay of evolved strain 13 is depicted above. (B) Growth assay confirming enhanced growth of a rnt1Δ ydr514cΔ double mutant compared to rnt1Δ. Experiment was performed using two independent biological replicates. (C) Growth assay confirming impaired growth of wild type and rnt1Δ strains harboring plasmids that overexpress wild-type YDR514C or the ydr514c stem loop mutant. Experiment was performed using two independent biological replicates. Strains were spotted on SC-Leu.
Bioinformatic pipeline for analysis of PARE data in Galaxy.
Pink boxes represent files used only for a subsequent step in the pipeline. Green boxes represent files used for further analysis in Microsoft Excel or Integrative Genomics Viewer (IGV).
Rnt1 cleavage site distribution.
496 Rnt1 cleavage sites are clustered into 166 putative substrates. Hits separated by 1 nt are likely due to residual Rat1 and/or Dxo1 activity, and hits separated by 20-60 nts are likely due to Rnt1 cutting both sides of a stem loop.
(A) Examples of known Rnt1 cleavage sites detected by PARE in ncRNAs: pre-rRNA 3’ ETS, pre-U1 snRNA, and pre-SNR190/128 snoRNA dicistronic transcript. (B) Novel Rnt1 cleavage sites detected by PARE at the 5’ ends of snoRNAs SNR39B, SNR85, SNR87, and SNR81. (C) Novel Rnt1 cleavage sites detected by PARE at the 3’ end of the U3 snRNA (SNR17B). (D) PARE detects a Rnt1 cleavage site at the mature 5’ end of SNR84 as well as sites 26 and 74 nts upstream of the mature end, suggesting Rnt1 cleavage of a 5’ extension upstream of pre-SNR84. (E) Example of an intron lacking a snoRNA that is still cleaved by Rnt1. (F-H) Rnt1 cleavage sites detected by PARE in intergenic regions: (F) between FRD1 and GLY1 contain a structural ncRNA; (G) between SKN1 and THI4 contain the uncharacterized ncRNA chrVII-0170-W; (H) between GCG1 and CHD1 contain the uncharacterized ncRNA chrV-0121-C. Probing the GCG1/CHD1 intergenic region by northern blot showed RNA stabilization in the absence of a catalytically active Rnt1. Red arrowheads, Rnt1 cleavage sites.
(A) ARN2 is a published mRNA target detected by PARE. Other newly identified and highly cleaved Rnt1 mRNA targets detected by PARE include TCB1, YER145C-A, PAN6, and AVT1. (B) The predicted secondary structures of novel Rnt1 mRNA targets in (A), with Rnt1 cleavage sites indicated by red arrowheads. (C) Rnt1 cleavage site detected in YPL277C but not in YOR389W presumably because of a single nt difference in the YPL277C stem loop that stabilizes the structure.
(A) Rnt1-cleaved mRNAs encode proteins with varying subcellular localization. (B) Rnt1-cleaved mRNAs encode proteins that carry out various cellular functions.
Predicted mRNA tetraloop sequences plus surrounding sequences used for alignment in Figure 2F.
(A) The rnt1-ΔNLS and rnt1-K45I mutants grow similarly to a strain containing wild-type RNT1 expressed from a plasmid. (B) YDR514C is cleaved more efficiently in the rnt1-ΔNLS strain compared to RNT1. (C) Rnt1 ncRNA targets SNR83, SNR190/128, and SNR81 are cleaved more efficiently in the rnt1-K45I mutant compared to the RNT1 strain, and less efficiently in the rnt1-ΔNLS strain compared to RNT1. (D) Although MIG2 is cleaved in in vitro, no PARE peaks are detected for in vivo cleavage, even in the rnt1-ΔNLS strain.
Rnt1 preferentially cleaves polyadenylated mRNA.
(A) Rnt1 cleavage products predominate over decapping products in poly(A)+ PARE. For each mRNA substrate identified as an Rnt1 target, the fraction of the degradome generated by Rnt1 was calculated as the frequency of Rnt1 cleavage products divided by the total amount of products resulting from both Rnt1 and decapping. These values were calculated from PARE peak height and represent the averages of two biological replicates. (B) NET-seq, which sequences mRNA 3’ OH ends still associated with RNA polymerase II, identifies the spliceosome-mediated decay (SMD) product of BDF2 (purple arrowhead) but does not identify prominent peaks corresponding to Rnt1 cleavage (red arrowheads), suggesting that Rnt1 cleaves after cleavage and polyadenylation and release from RNA polymerase II. Grey bars, decapping peaks.
Rnt1 PARE data generated in this study (top two panels), compared to one replicate of RNT1 vs rnt1Δ RNA-seq data previously published by Grzechnik et al., 2018 (bottom two panels).
Rnt1 may affect the gene expression levels of some mRNA targets such as CAF4, YDR514C, and MTM1, but not of others like BDF2.
(A) Evolved rnt1Δ strains grow better than the rnt1Δ parent strain at both 30 °C and 37 °C. Mutations identified by whole-genome sequencing are shown on the right. (B) AlphaFold-predicted structure of the Ydr514c nuclease domain (left) and putative active site (right). Red, oxygens; blue, nitrogen; cyan, glycine 220 which was mutated to serine in evolved rnt1Δ strain 13.
List of plasmids used in this study:
List of yeast strains used in this study:
