Nociceptors located in the TG and DRG are the primary sensors of damaging or potentially damaging stimuli for the head and body, respectively, and are key drivers of chronic pain states. While nociceptors in these two tissues show a high degree of functional similarity, there are important differences in their development lineages, their functional connections to the central nervous system, and recent genome-wide analyses of gene expression suggest that they possess some unique genomic signatures. Here, we used translating ribosome affinity purification (TRAP) to comprehensively characterize and compare mRNA translation in Scn10a-positive nociceptors in the TG and DRG of male and female mice. This unbiased method independently confirms several findings of differences between TG and DRG nociceptors described in the literature but also suggests preferential utilization of key signaling pathways. Most prominently, we provide evidence that translational efficiency in mechanistic target of rapamycin (mTOR)-related genes is higher in the TG compared to DRG while several genes associated with the negative regulator of mTOR, AMPK activated protein kinase (AMPK), have higher translational efficiency in DRG nociceptors. Using capsaicin as a sensitizing stimulus we show that behavioral responses are greater in the TG region and this effect is completely reversible with mTOR inhibition. These findings have implications for the relative capacity of these nociceptors to be sensitized upon injury. Altogether, our data provide a comprehensive, comparative view of transcriptome and translatome activity in TG and DRG nociceptors that enhances our understanding of nociceptor biology.

SIGNIFICANCE STATEMENT

The DRG and TG provide sensory information from the body and head, respectively. Nociceptors in these tissues are critical first neurons in the pain pathway. Injury to peripheral neurons in these tissues can cause chronic pain. Interestingly, clinical and preclinical findings support the conclusion that injury to TG neurons is more likely to cause chronic pain and chronic pain in the TG area is more intense and more difficult to treat. We used TRAP technology to gain new insight into potential differences in the translatomes of DRG and TG neurons. Our findings demonstrate previously unrecognized differences between TG and DRG nociceptors that provide new insight into how injury may differentially drive plasticity states in nociceptors in these two tissues.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina

TET enzymes convert 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized derivatives. TETs stably associate with and are post-translationally modified by the nutrient-sensing enzyme OGT, suggesting a connection between metabolism and the epigenome. Here, we show for the first time that modification by OGT enhances TET1 activity in vitro. We identify a TET1 domain that is necessary and sufficient for binding to OGT and report a point mutation that disrupts the TET1-OGT interaction. We show that this interaction is necessary for TET1 to rescue hematopoetic stem cell production in tet mutant zebrafish embryos, suggesting that OGT promotes TET1’s function during development. Finally, we show that disrupting the TET1-OGT interaction in mouse embryonic stem cells changes the abundance of TET2 and 5-methylcytosine, which is accompanied by alterations in gene expression. These results link metabolism and epigenetic control, which may be relevant to the developmental and disease processes regulated by these two enzymes.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat) and SENSE Total RNA-Seq Library Prep Kit

The oncofetal mRNA-binding protein IGF2BP1 and the transcriptional regulator SRF modulate gene expression in cancer. In cancer cells, we demonstrate that IGF2BP1 promotes the expression of SRF in a conserved and N⁶-methyladenosine (m⁶A)-dependent manner by impairing the miRNA-directed decay of the SRF mRNA. This results in enhanced SRF-dependent transcriptional activity and promotes tumor cell growth and invasion. At the post-transcriptional level, IGF2BP1 sustains the expression of various SRF-target genes. The majority of these SRF/IGF2BP1-enhanced genes, including PDLIM7 and FOXK1, show conserved upregulation with SRF and IGF2BP1 synthesis in cancer. PDLIM7 and FOXK1 promote tumor cell growth and were reported to enhance cell invasion. Consistently, 35 SRF/IGF2BP1-dependent genes showing conserved association with SRF and IGF2BP1 expression indicate a poor overall survival probability in ovarian, liver and lung cancer. In conclusion, these findings identify the SRF/IGF2BP1-, miRNome- and m⁶A-dependent control of gene expression as a conserved oncogenic driver network in cancer.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression. The effect of forced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we conducted artificial methylation of thousands of promoters in human cells using an artificial zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription and histone modifications, and the durability of DNA methylation after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)

eIF5A is an essential protein involved in protein synthesis, cell proliferation and animal development. High eIF5A expression is observed in many tumor types and has been linked to cancer metastasis. Recent studies have shown that eIF5A facilitates the translation elongation of stretches of consecutive prolines. Activated eIF5A binds to the empty E-site of stalled ribosomes, where it is thought to interact with the peptidyl-tRNA situated at the P-site. Here, we report a genome-wide analysis of ribosome stalling in Saccharomyces cerevisiae eIF5A depleted cells using 5Pseq. We confirm that, in the absence of eIF5A, ribosomes stall at proline stretches, and extend previous studies by identifying eIF5A-dependent ribosome pauses at termination and at >200 tripeptide motifs. We show that presence of proline, glycine and charged amino acids at the peptidyl transferase center and at the beginning of the peptide exit tunnel arrest ribosomes in eIF5A-depleted cells. Lack of eIF5A also renders ribosome accumulation at the stop codons. Our data indicate specific protein functional groups under the control of eIF5A, including ER-coupled translation and GTPases in yeast and cytoskeleton organization, collagen metabolism and cell differentiation in humans. Our results support a broad mRNA-specific role of eIF5A in translation and identify the conserved motifs that affect translation elongation from yeast to humans.

Features RiboCop rRNA Depletion Kit V1.2 (Human/Mouse/Rat)