Splicing inactivation generates hybrid mRNA-snoRNA transcripts targeted by cytoplasmic RNA decay

Significance Small nucleolar RNAs (snoRNAs) mediate modifications of nucleosides within ribosomal RNAs, which are necessary for proper ribosomal function and translation. Many snoRNAs are encoded within introns of host genes, and accurate biogenesis of these small RNAs is required to produce functional snoRNAs. The work presented here shows that when the splicing reactions are inactivated, snoRNAs undergo a distinct biogenesis pathway, which leads to the production of aberrant hybrid RNAs that contain both messenger RNA (mRNA) and small RNA components of the host genes. While snoRNAs are primarily found in the nucleolus, these hybrid RNAs are degraded by the cytoplasmic mRNA degradation pathway. These results demonstrate the importance of splicing to promote accurate snoRNA processing and prevent the production of aberrant mRNA-snoRNA hybrids.


Supporting Materials and Methods
Oxford Nanopore Sequencing The Saccharomyces cerevisiae Slu7p anchor-away strains was used as a source of RNA. The strain was grown in YPD and when OD600 reached 0.5, Slu7p was depleted from the nucleus by addition of rapamycin to a final concentration of 1ug/ml and incubated for 1 hour. 40 ug of total RNAs extracted from these cells were treated with DNAse I (Invitrogen, catalog #: 18-068-015) according to the manufacturer's protocol. DNase I treated RNAs were incubated with Shrimp Alkaline Phosphatase (NEB, catalog #: M0371S) in order to remove 3' phosphates. This was followed up by treatment with Terminator™ exonuclease (Lucigen, catalog #: MA246E) digestion, as specified in the manufacturer's protocol, to degrade RNAs with 5' monophosphates. The remaining RNAs were then in vitro polyadenylated using E. coli Poly(A) Polymerase (NEB, catalog #: M0276S). Phenol chloroform extraction and ethanol precipitation was performed after each enzymatic treatment.
RNA libraries were prepared from 500 ng of in vitro polyadenylated RNAs using the direct RNA sequencing kit from Oxford Nanopore (ONT, catalog #: SQK-RNA002) as per the manufacturer's instructions. Sequencing was performed using R9.4 flow cells on a MinION Mk1B device and sequenced for 48 hours. Basecalling was performed using Guppy Basecaller (Version 6.1.1+1f6bfa7f8). Reads were then mapped to the Saccharomyces cerevisiae genome (S288C_reference_sequence_R64-3-1) using Minimap 2 (Version 2.17-r941). Reads were visualized using IGV (Version 2.12.3) and figures were prepared using Inkscape (Version 1.1.2).
Data is available as unprocessed FAST5 files, FASTQ files and aligned bam files at NCBI as BioProject ID: PRJNA827814.

RNA immunoprecipitation
Yeast cells corresponding to 400 OD600 units of culture were harvested in exponential phase, washed and resuspended in lysis buffer (20mM Tris-HCl pH=8, 300mM K-Acetate, 5mM MgCl2, 1mM Dithiothreitol, 0.2% Triton X-100, protease inhibitors cocktail tablet (Roche) ). Cells were lysed by vortexing in the presence of glass beads (425-600 μm) for 5 minutes. Whole cell lysates were collected after centrifugation at 15,000 rpm for 20 minutes. About 1000μL of lysate and 50μL of pre-washed mouse IgG conjugated magnetic beads (Cell Signaling) were incubated for one hour on a shaker at 4℃. After purification of the beads on a magnetic rack, beads were washed five times with 1000μL of lysis buffer and RNA extraction after the final wash were performed using phenol: chloroform: iso-amyl alcohol solution (VWR). Figure S1. Analysis of hmsnoRNA 3' ends using 3' RACE (A) Schematic of 3' RACE procedure. RNA with an intron-encoded snoRNA is depicted. The RNA was in vitro polyadenylated and an oligo d(T)-containing primer (prMG1) was annealed to the poly-A tail (top panel). RNA was reverse transcribed to cDNA and amplified by PCR (lower panel). Primers prMG3 and prMG5 anneal to the junction between exon 1 and the upstream intron while primers prMG4 and prMG6 anneal to the intron. Primer prMG2 anneals to the 5' sequence of prMG1, added to the 3' end of cDNA during reverse transcription.

Supporting Figures
(B-C) Sequences of the amplified 3' ends of hmsnoRNAs from NOG2/snR191 (B) and IMD4/snR54 (C) obtained from RNAs extracted from a Slu7p-anchor away strain post-rapamycin treatment. Sequences aligning to the snoRNA are bolded, and the poly-A sequence added by in vitro polyadenylation is italicized. A. Structure of the ASC1/snR24 gene and location of the probes used for differential northern blot analysis in panel B. Legends as in Figure 1A. Boxes and line lengths are not to scale.

B.
Northern blot analysis of ASC1/snR24 in the Slu7p-AA strain using probes hybridizing to the indicated regions of the ASC1/snR24 gene. An ethidium bromide staining of the 25S rRNA is shown as a loading control. The ASC1-snR24 hmsnoRNA species were detected by the 5′-exon and snoRNA probes (probes 1 and 3), and by an intronic probe located 5′ to the snoRNA (probe 2), but not to a probe hybridizing to the 3′-exon (probe 4). As expected, probe 1 also detected the cleaved 5′-exon intermediate, and C. Analysis of the polyadenylation status of ASC1/snR24. Shown is a northern blot of ASC1/snR24 using a 5′exon probe (#1 in Figure 2C) of RNAs extracted from the Slu7p-FRB tagged strain grown after treatment with rapamycin. T = total RNAs; pA+= polyadenylated RNAs selected by oligodT affinity; pA-= Non-polyadenylated RNAs extracted from the supernatant of the oligodT affinity purification. scR1 was used as a non-polyadenylated RNA negative control. B. RPS22B Region. Figure S4. Northern blot analysis of NOG2/snR191 using a probe complementary to the exon1 of NOG2 or a probe complementary to snR191 in strains expressing anchor-away versions of Rrp6p, Slu7p, or both Slu7p and Rrp6p. Strains in the name of the gene italicized indicate that the corresponding protein was FRB-tagged (eg rrp6 = rrp6-FRB). Each FRB tagged strain was grown in normal medium (-Rapa) or shifted for 1hr in a medium containing Rapamycin (+Rapa) to promote export of the corresponding proteins out of the nucleus. GAPDH was used as a loading control. Labeling of the species as in Main Figure  1. scR1 was used as a loading control. Figure S5. Northern blot analysis of NOG2 in a wild-type strain transformed with a centromeric vector (pUG35) or pUG35 containing a NOG2 gene insert.
The NOG2 gene inserted in the pUG35 plasmid is expressed under the control of its endogenous promoter. The top panel shows a northern blot analysis of NOG2 using a probe hybridizing to the exon1 of NOG2. The bottom panel shows an Ethidium bromide staining of rRNAs from the gel used for the northern blot shown above. The NOG2 label indicates the spliced NOG2 mRNA.    Figure 4C vs. the RNAs detected in the input fractions.
Ratios are shown for the two independent biological replicates. The scR1 RNA was not analyzed for Replicate 1.  Shown are northern blots of NOG2 of RNAs extracted from wild-type yeast strain grown in log phase (log), stationary phase (stat), exposed for 2hours of heat shock (39°C HS) or treated for one hour with Rapamycin. None of the conditions used resulted in the production of hmsnoRNAs. Only the spliced form of NOG2 (S) is indicated.