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Chemoproteomic discovery of a human RNA ligase

ABSTRACT: RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5'-PO4 and 3'-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as the first human enzyme promoting RNA ligation between 5'-PO4 and 3'-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. This data provides the groundwork for establishing a human RNA repair pathway.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Philip Saumer

LAB HEAD: Andreas Marx

PROVIDER: PXD038132 | Pride | 2023-01-27

REPOSITORIES: Pride

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			Action	DRS
	H1299_MaxQuant.zip	Other
	H1299_Q1904_hoellmuellere_163.raw	Raw
	H1299_Q1904_hoellmuellere_164.raw	Raw
	H1299_Q1904_hoellmuellere_166.raw	Raw
	H1299_Q1904_hoellmuellere_167.raw	Raw

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Project description:2×10^7 HL60 cells were treated with 10 uM ATRA for 2 and 5 days, and conducted Hi-C to study the chromosomal architecture. Cells were fixed by 1% formaldehyde (v/v) for 15 min at room temperature (RT) with slowly rotation. Cells were lysed by 550 μl lysis buffer (500 μl 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630 and 50 μl protease inhibitors) using a homogenizer, and harvested the chromatin at 5,000 rpm and washed twice. The pellet was added 1% SDS and incubated at 65 oC for 10 min, then quenched by Triton X-100. The chromatin was digested by 600 U MboI (NEB, Ipswich, USA) at 37 oC overnight. The next day, enzymes were inactivated by 10% SDS at 65 oC for 30 min, and the restriction fragment overhangs were filled in using biotin-14-dCTP and Klenow at 37 oC for 45 min, and the ligation was conducted in 7.61 ml ligation mix (745 μl 10% Triton X-100, 745 μl 10x ligation buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 100 mM DTT), 80 μl 10 mg/ml BSA, 80 μl 100 mM ATP and 5.96 ml water) for 4 h at 16°C. The blunt-end Hi-C ligation was sonicated and pull down by streptavidin beads, then purified by ethanol. Hi-C library was prepared for paired ending sequencing using NEBNext® UltraTM DNA Library Prep Kit for Illuina® (NEB). For regular 3C ligation, after enzymes inactivation, 10 μl 1 U/μl T4 DNA ligase was added to incubate at 16°C overnight. The interaction was detected by qPCR using the specific 3C primers (Table 1) followed by DNA purification. Hi-C was conducted in PE-150. An iterative method for Hi-C mapping with initial length 30bp, if it fails to map uniquely, the next round of mapping for additional 20 bp continued, this procedure lasts until the full read length reaches 150bp. The Hi-C reads were mapped to the human reference genome (assembly GRCh38) using bowtie2 (v2.2.9). The reads mapped to the same restriction fragment, the reads less than 500bp and PCR duplicates were all removed. Hi-C contact maps were normalized using ICE.

Project description:Purpose: Ribosome profiling and RNA-Seq were used to map the location and abundance of translating ribosomes on human skeletal muscle transcripts from a patient with Becker muscular dystrophy. Methods: Tissue homogenates were prepared from frozen sections of a muscle biopsy obtained from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control. Ribosome-protected fragments and total RNA were prepared from a single homogenate, so starting RNA populations for both libraries were closely matched. Homogenates were not clarified before RNase digestion to avoid loss of ribosomes associated with large molecular weight complexes and RNA-Seq libraries were prepared after rRNA subtraction to avoid positional loss of 5’ reads. RPF-Seq libraries were built using the TruSeq Small RNA Sample Preparation Kit (Illumina) and RNA-Seq libraries were built using the TruSeq RNA Sample Preparation v2 Kit (Illumina). RPF-Seq and RNA-Seq libraries were subjected to 50 cycles of single-end sequencing on an Illumina HiSeq 2000 instrument. Trimmed and filtered RPF- and RNA-Seq reads were mapped to RefSeq fasta sequences downloaded from the UCSC genome browser (hg19 assembly). Results: Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that results in the expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosomal profiling. Conclusions: Our results provide a molecular explanation for the rescue of 5’ truncating mutations via a heretofore undescribed mechanism of post-transcriptional regulation of dystrophin expression. The presence of a glucocorticoid-inducible IRES within a highly conserved region of the DMD sequence strongly suggests a programmed role for alternate translation initiation, and ongoing efforts to understand the relevant cell lineage-specific and/or conditional activation signals will shed light on underlying mechanisms of IRES control and elucidate potentially novel functions of dystrophin. Skeletal muscle ribosome-protected fragment and RNA-Seq profiles from a patient with an NM_004006:c.40_41delGA dystrophin mutation and a normal control were generated by deep sequencing using the Illumina HiSeq 2000.

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Chemoproteomic discovery of a human RNA ligase

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