RRNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilize the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase center and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2′-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity. While the mechanisms regulating partial modification and the functions of specialized ribosomes are largely unknown, changes in the rRNA modification pattern have been observed in response to environmental changes, during development, and in disease. This suggests that rRNA modifications may contribute to the translational control of gene expression.

5-methylcytosine (m⁵C) is an abundant RNA modification that’s presence is reported in a wide variety of RNA species, including cytoplasmic and mitochondrial ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs), as well as messenger RNAs (mRNAs), enhancer RNAs (eRNAs) and a number of non-coding RNAs. In eukaryotes, C5 methylation of RNA cytosines is catalyzed by enzymes of the NOL1/NOP2/SUN domain (NSUN) family, as well as the DNA methyltransferase homologue DNMT2. In recent years, substrate RNAs and modification target nucleotides for each of these methyltransferases have been identified, and structural and biochemical analyses have provided the first insights into how each of these enzymes achieves target specificity. Functional characterizations of these proteins and the modifications they install have revealed important roles in diverse aspects of both mitochondrial and nuclear gene expression. Importantly, this knowledge has enabled a better understanding of the molecular basis of a number of diseases caused by mutations in the genes encoding m⁵C methyltransferases or changes in the expression level of these enzymes.

Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production.

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N⁶-methyladenosine (m⁶A) modifications in RNAs play important roles in regulating many different aspects of gene expression. While m⁶As can have direct effects on the structure, maturation, or translation of mRNAs, such modifications can also influence the fate of RNAs via proteins termed “readers” that specifically recognize and bind modified nucleotides. Several YTH domain-containing proteins have been identified as m⁶A readers that regulate the splicing, translation, or stability of specific mRNAs. In contrast to the other YTH domain-containing proteins, YTHDC2 has several defined domains and here, we have analyzed the contribution of these domains to the RNA and protein interactions of YTHDC2. The YTH domain of YTHDC2 preferentially binds m⁶A-containing RNAs via a conserved hydrophobic pocket, whereas the ankyrin repeats mediate an RNA-independent interaction with the 5′–3′ exoribonuclease XRN1. We show that the YTH and R3H domains contribute to the binding of YTHDC2 to cellular RNAs, and using crosslinking and analysis of cDNA (CRAC), we reveal that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites. YTHDC2 was recently found to promote a “fast-track” expression program for specific mRNAs, and our data suggest that YTHDC2 accomplishes this by recruitment of the RNA degradation machinery to regulate the stability of m⁶A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m⁶A-containing mRNAs and the ribosomes to facilitate their efficient translation.