GSM2840017: WT36_RiboSeq; Mus musculus; OTHER - SRA

SRX3362056: GSM2840017: WT36_RiboSeq; Mus musculus; OTHER
3 ILLUMINA (Illumina HiSeq 2500) runs: 185.2M spots, 9.8G bases, 3.7Gb downloads

Submitted by: NCBI (GEO)

Study: Translatome analysis of the ribosomal protein L10 R98S mutation reveals altered serine metabolism in acute lymphoblastic leukemia [Ribo-seq]

PRJNA417116 • SRP123624 • All experiments • All runs

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Somatic ribosomal protein defects have recently been described in cancer, yet their impact on cellular transcription and translation remain poorly understood. Here we integrated mRNA sequencing, ribosome footprinting, polysomal RNA seq and quantitative mass spectrometry datasets obtained from an isogenic mouse lymphoid cell model in order to study the T-cell acute lymphoblastic leukemia (T-ALL) associated R98S mutation in ribosomal protein L10 (RPL10 R98S). RPL10 R98S induced changes in protein levels were to a much larger extent caused by transcriptional then translational changes and RPL10 R98S cells showed a gene signature corresponding to deregulation of hematopoietic transcription factors. Phosphoserine phosphatase (PSPH), a key enzyme in serine biosynthesis, displayed elevated transcription and translation and was one of the proteins showing the strongest upregulation in RPL10 R98S cells. Increased Psph protein levels were confirmed in RPL10 R98S engineered JURKAT cells and in hematopoietic cell cultures derived from Rpl10 R98S knock-in mice. Moreover, elevated serine and glycine biosynthesis in RPL10 R98S cells was supported by metabolic flux analyses. Analysis of PSPH expression levels in T-ALL patient samples revealed that PSPH upregulation is a generalized phenomenon in this disease, associated with elevated circulating serine and glycine levels. Addition of serine and glycine enhanced survival of stromal and myeloid cells, suggesting supportive effects on the hematopoietic niche. Finally, reduction of PSPH expression levels in T-ALL cell lines suppressed their in vitro proliferation and their capacity to expand in T-ALL xenograft models. In conclusion, transcriptome, translatome and proteome analysis of the RPL10 R98S mutation identified RPL10 R98S driven induction of cellular serine biosynthesis. Whereas serine metabolism has been implicated in cancer via PHGDH amplification, this is the first report supporting dependence of ALL cells on the serine biosynthesis enzyme PSPH. Overall design: Ribosome footprinting and mRNA-Seq libraries from 3 biological replicates for each condition (RPL10 R98S, RPL10 WT). For ribosome footprinting three technical replicates were generated for each biological replicate. The sequencing files of three technical replicates were merged for downstream analyses

Sample: WT36_RiboSeq

SAMN07977214 • SRS2660739 • All experiments • All runs

Organism: Mus musculus

Library:

Instrument: Illumina HiSeq 2500

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: SINGLE

Construction protocol: Ribosome footprinting libraries. RNA fragments were purified using Trizol. RNA-Seq libraries. RNA was isolated using the Maxwell 16 LEV simplyRNA Cells Kit (Promega) Ribosome footprinting libraries. RNA was gel-purified on a denaturing 10% polyacrylamide urea (7 M) gel. A section corresponding to 30 to 33 nucleotides was excised, eluted and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 PNK (New England Biolabs) for 4 hrs at 37°C in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (New England Biolabs) for 2.5 hrs at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 hrs at 22°C. To deplete ribosomal RNAs (rRNA), the resulting fragments were incubated with a biotinylated rRNAs oligo pool (10 μM each) for 10 min at 37°C, upon short denaturation at 95-100 °C for 1 min. rRNA depletion was carried on using MyOne Streptavidin C1 DynaBeads (ThermoFisher Scientific). The resulting rRNA-depleted fragments were reverse transcribed using the SuperScript III cDNA synthesis kit. Samples were PCR-amplified for 15 cycles and the products were multiplexed and sequenced on an Illumina HiSeq 2000 platform in single read 50 nucleotides mode. RNA-Seq libraries. Libraries were generated from total RNA using the TruSeq Stranded mRNA Sample Prep Kit (Illumina)

Experiment attributes:

GEO Accession: GSM2840017

Links:

NCBI link: NCBI Entrez (gds)

Runs: 3 runs, 185.2M spots, 9.8G bases, 3.7Gb

Run	# of Spots	# of Bases	Size	Published
SRR6255575	36,899,220	2.2G	658.9Mb	2019-04-10
SRR6255576	37,708,039	1.9G	629.4Mb	2019-04-10
SRR6255577	110,629,054	5.6G	2.5Gb	2019-04-10

ID:: 4696498

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