DOI: 10.5061/dryad.5tb2rbp8s
title: Intestinal transit amplifying cells require METTL3 for growth factor signaling and cell survival

description: Intestinal epithelial transit amplifying cells are essential stem
 progenitors required for intestinal homeostasis, but their rapid
 proliferation renders them vulnerable to DNA damage from radiation and
 chemotherapy. Despite their critical roles in intestinal homeostasis and
 disease, few studies have described genes that are essential to
 transit-amplifying cell function. We report that the RNA
 methyltransferase, METTL3, is required for survival of transit-amplifying
 cells in the murine small intestine. Transit-amplifying cell death after
 METTL3 deletion was associated with crypt and villus atrophy, loss of
 absorptive enterocytes, and uniform wasting and death in METTL3-depleted
 mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in
 vivo demonstrated decreased translation of hundreds of unique methylated
 transcripts after METTL3 deletion, particularly transcripts involved in
 growth factor signal transduction such as Kras. Further investigation
 confirmed a relationship between METTL3 and Kras methylation and protein
 levels in vivo. Our study identifies METTL3 as an essential factor
 supporting the homeostasis of small intestinal tissue via direct
 maintenance of transit-amplifying cell survival. We highlight the crucial
 role of RNA modifications in regulating growth factor signaling in the
 intestine, with important implications for both homeostatic tissue renewal
 and epithelial regeneration.
description: These data were processed using open sourced software as
 described in the methods section 
description: # RNAseq, Polysomeseq, and m6aseq in mouse small intestinal epithelium
 This dataset contains the results of three sequencing experiments: RNAseq,
 Polysome-seq, m6Aseq.  A summary of experimental procedures is included
 below. For full methods details please refer to the associated research
 article.    ### RNAseq Mettl3-flox/flox and VillinCreER;Mettl3-flox/flox
 ileal enteroids were expanded in Matrigel in 50% WRN media until
 approximately 8 to 10 million cells per replicate was achieved.  For each
 genotype, 3 passage separated replicates from the same enteroid line were
 used.  Media was supplemented with 2 uM 4-OHT to induce METTL3 deletion 72
 hours prior to collection.  Libraries were prepared from total RNA using
 the Smarter® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara
 Bio 634411) and sequenced on a Novaseq 6000, SP Reagent Kit 1.5 (100
 cycles). Libraries were demultiplexed with Illumina DRAGEN. The reads were
 aligned to all transcripts on the mouse reference chromosomes (Gencode
 version M31, GRCm39) using Kallisto. ### Polysome-seq (note that
 polysome-seq files have "riboseq" prefix") Mettl3-flox/flox
 and VillinCreER;Mettl3-flox/flox ileal enteroids were expanded in Matrigel
 in 50% WRN media until approximately 8 to 10 million cells per replicate
 was achieved.  For each genotype, 3 passage separated replicates from the
 same enteroid line were used.  Media was supplemented with 2 uM 4-OHT to
 induce METTL3 deletion 72 hours prior to collection.  Polysomal RNAs were
 isolated as described in:  A. L. Cope, S. Vellappan, J. S. Favate, K. S.
 Skalenko, S. S. Yadavalli, P. Shah, Exploring Ribosome-Positioning on
 Translating Transcripts with Ribosome Profiling. Methods Mol. Biol. 2404,
 83–110 (2022). Once the rRNA-depleted polysomal RNA was obtained, it was
 pooled together, and the libraries were prepared.  The final library
 contains a 5' adapter – 4 random bases – insert – 5 random bases –
 sample barcode – 3’ adapter.  The randomized bases function as Unique
 molecular identifiers (UMIs) for deduplication.  The multiplexed library
 was then sequenced on Illumina HiSeq 4000 with PE150 runs (paired-end
 reading of 150 bases), with a sequencing depth of 60 million raw reads per
 sample.  For computational analysis, the raw sequencing data were
 demultiplexed, the adaptors were removed using cutadapt, the contaminant
 sequences (rRNA and tRNA) were depleted, and the reads were deduplicated
 (umi\_tools dedup).  The reads were aligned to all transcripts on the
 mouse reference chromosomes (Gencode version M31, GRCm39) using Kallisto.
 Translation efficiency (TE) was calculated by dividing the TPM in the
 total RNA library by the TPM in the polysome-seq library for each
 individual transcript and sample. ### m6Aseq Three wildtype C57BL/6J mice
 (Jax #000664, 2 male 1 female) aged 8 weeks were used.  Intestinal crypts
 were isolated from the distal half of the small intestine.  Isolated
 crypts were dissociated to single cells, epithelial cells were stained
 with fluorescent antibodies marking epithelial cells.  Approximately 800K
 live epithelial cells per mouse were then isolated by flow cytometry.
 RIP-seq was performed according to the “Refined RIP-seq” protocol for low
 input material. In brief, 5% of total RNA was set aside as input,
 remaining RNA was immunoprecipitated twice in succession with two
 independent m6a-specific antibodies. Final cDNA Libraries were prepared
 from twice immunoprecipitated RNA and fragmented input total RNA using the
 Smarter® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio
 634411) and sequenced on a Novaseq 6000, SP Reagent Kit 1.5 (100 cycles). 
 Raw reads were aligned to mm10 (gencode\_M23\_GRCm38.p6) using STAR
 aligner (2.7.9a).  N6-methyladenosine peaks were identified using
 exomePeak2 (version 1.2.0) ## Description of the data and file structure
 Fastq files are provided. Files are demultiplexed but aligned and
 unfiltered. The pipeline used for mapping and analysis is described above.
 ### RNAseq Files with prefix "C" are CTRL mettl3-flox/flox
 samples Files with prefix "KO" are METTL3 KO
 vilcreert2;mettl3-flox/flox samples ### Polysomeseq Files with prefix
 "Riboseq\_CTRL" are CTRL mettl3-flox/flox samples Files with
 prefix "Riboseq\_METTL3KO" are METTL3 KO
 vilcreert2;mettl3-flox/flox samples ### m6Aseq Files with prefix ending in
 "i" are input RNA Files wih prefix ending in "r" are
 m6A-RIP RNA Prefix "M2" and "M3" refer to male mouse
 replicates Prefix "F3" refers to female mouse replicates