The three 'nonsense' or 'stop' codons that signal the ribosome to cease translation — UAA, UAG and UGA — all contain an initial uridine residue. John Karijolich and Yi-Tao Yu demonstrate that isomerization of this uridine, converting it to pseudouridine (Ψ), allows the codons to be read by the ribosome in vivo. Specific amino acids are inserted into the resulting protein, with ΨAA and ΨAG coding for serine and threonine, and ΨGA for tyrosine and phenylalanine. Although there is yet no evidence for a physiological role for this RNA modification, it could offer a way of expanding the genetic code. Similar RNA modification could also be useful as a means of nonsense suppression in vivo, either experimentally or clinically. All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon1,2,3. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.

Spliceosomal snRNAs are extensively 2'-O-methylated and pseudouridylated. The modified nucleotides are relatively highly conserved across species, and are often clustered in regions of functional i...

Background: Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown. Methods: The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining. Results: The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/β-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (m⁶A) site of TCF7L2 mRNA in the presence of MIR100HG. TCF7L2, in turn, activated MIR100HG transcription, forming a feed forward regulatory loop. The MIR100HG/hnRNPA2B1/TCF7L2 axis was augmented in specimens from CRC patients who either developed local or distant metastasis or had disease progression that was associated with cetuximab resistance. Conclusions: MIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Methylation at the N⁶ position of adenosine (m⁶A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m⁶A deposition and proteins involved in the m⁶A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m⁶A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m⁶A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m⁶A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m⁶A levels across much of the host transcriptome. However, we found that depletion of the m⁶A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m⁶A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m⁶A modified. In contrast, although the ORF50 mRNA was also m⁶A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m⁶A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types. In addition to its roles in regulating cellular RNA fate, methylation at the N⁶ position of adenosine (m⁶A) of mRNA has recently emerged as a mechanism for regulating viral infection. While it has been known for over 40 years that the mRNA of nuclear replicating DNA viruses contain m⁶A, only recently have studies began to examine the distribution of this modification across viral transcripts, as well as characterize its functional impact upon viral lifecycles. Here, we apply m⁶A-sequencing to map the location of m⁶A modifications throughout the transcriptome of the oncogenic human DNA virus Kaposi’s sarcoma-associated herpesvirus (KSHV). We show that the m⁶A machinery functions in a cell type specific manner to either promote or inhibit KSHV gene expression. Thus, the KSHV lifecycle is impacted by the m⁶A pathway, but the functional outcome may depend on cell lineage specific differences in m⁶A-based regulation.