One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc–siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc–siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.

SUMMARY Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We identified an abundantly-expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly-conserved sequence similar to the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1’s conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival as compared to females.

The Saccharomyces cerevisiae gene YPK9 encodes a putative integral membrane protein which is 58% similar and 38% identical in amino acid sequence to the human lysosomal P5B ATPase ATP13A2. Mutations in ATP13A2 have been found in patients with Kufor-Rakeb syndrome, a form of juvenile Parkinsonism. We report that Ypk9p localizes to the yeast vacuole and that deletion of YPK9 confers sensitivity for growth for cadmium, manganese, nickel or selenium. These results suggest that Ypk9p may play a role in sequestration of divalent heavy metal ions. Further studies on the function of Ypk9p/ATP13A2 may help to define the molecular basis of Kufor-Rakeb syndrome and provide a potential link to environmental factors such as heavy metals contributing to some forms of Parkinsonism.

The advent of high throughput sequencing technologies has revealed that pervasive transcription generates RNAs from nearly all regions of eukaryotic genomes. Normally these transcripts undergo rapid degradation by a nuclear RNA surveillance system primarily featuring the RNA exosome. This multimeric protein complex plays a critical role in the efficient turnover and processing of a vast array of RNAs in the nucleus. Despite its initial discovery over a decade ago, important questions remain concerning the mechanisms that recruit and activate the nuclear exosome. Specificity and modulation of exosome activity requires additional protein cofactors, including the conserved TRAMP polyadenylation complex. Recent studies suggest that helicase and RNA binding subunits of TRAMP direct RNA substrates for polyadenylation, which enhances their degradation by Dis3/Rrp44 and Rrp6, the two exosome associated ribonucleases. These findings indicate that the exosome and TRAMP have evolved highly flexible functions that allow recognition of a wide range of RNA substrates. This flexibility provides the nuclear RNA surveillance system with the ability to regulate the levels of a broad range of coding and non-coding RNAs, which results in profound effects on gene expression, cellular development, gene silencing and heterochromatin formation. This review summarizes recent findings on the nuclear RNA surveillance complexes, and speculates upon possible mechanisms for TRAMP-mediated substrate recognition and exosome activation.

interfering ribonucleic acid; siRNA1-siRNA22, 22 distinct siRNAs used in the in vitro and in vivo studies with data presented in this work; STC, standard template chemistry; t 1/2 , half-life; T max , time at which maximal concentration was observed.