Oscillations are a key to achieving dynamic behavior and thus occur in biological systems as diverse as the beating heart, defecating worms, and nascent somites. Here we report pervasive, large-amplitude, and phase-locked oscillations of gene expression in developing C. elegans larvae, caused by periodic transcription. Nearly one fifth of detectably expressed transcripts oscillate with an 8 hr period, and hundreds change >10-fold. Oscillations are important for molting but occur in all phases, implying additional functions. Ribosome profiling reveals that periodic mRNA accumulation causes rhythmic translation, potentially facilitating transient protein accumulation as well as coordinated production of stable, complex structures such as the cuticle. Finally, large-amplitude oscillations in RNA sampled from whole worms indicate robust synchronization of gene expression programs across cells and tissues, suggesting that these oscillations will be a powerful new model to study coordinated gene expression in an animal.

Nonsense-mediated mRNA decay (NMD) is traditionally portrayed as a quality-control mechanism that degrades mRNAs with truncated open reading frames (ORFs). However, it is meanwhile clear that NMD also contributes to the post-transcriptional gene regulation of numerous physiological mRNAs. To identify endogenous NMD substrate mRNAs and analyze the features that render them sensitive to NMD, we performed transcriptome profiling of human cells depleted of the NMD factors UPF1, SMG6, or SMG7. It revealed that mRNAs up-regulated by NMD abrogation had a greater median 39-UTR length compared with that of the human mRNAome and were also enriched for 39-UTR introns and uORFs. Intriguingly, most mRNAs coding for NMD factors were among the NMD-sensitive transcripts, implying that the NMD process is autoregulated. These mRNAs all possess long 39 UTRs, and some of them harbor uORFs. Using reporter gene assays, we demonstrated that the long 39 UTRs of UPF1, SMG5, and SMG7 mRNAs are the main NMD-inducing features of these mRNAs, suggesting that long 39 UTRs might be a frequent trigger of NMD.

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing.