Summary The role of Dicer's helicase domain is enigmatic, but in vivo it is required for processing certain endogenous siRNA, but not miRNA. Using C. elegans extracts, or purified Drosophila Dicer-2, we compared activities of wildtype enzymes and those containing mutations in the helicase domain. We found the helicase domain was essential for cleaving dsRNA with blunt or 5' overhanging termini, but not those with 3' overhangs, as found on miRNA precursors. Further, blunt termini, but not 3' overhangs, led to increased siRNAs from internal regions of dsRNA; this activity required ATP and a functional helicase domain. Our data suggest that blunt or 5' overhanging termini engage Dicer's helicase domain to facilitate accumulation of siRNAs from internal regions of a dsRNA, an activity suited for processing long siRNA precursors of low abundance, but not necessary for the single cleavage required for miRNA processing.

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive doublestranded 39 UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 39 UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 39 UTRs are present on translating ribosomes.

HIV type 1 (HIV-1) envelope is a noncovalent trimer of gp120-gp41 heterodimers, and its lability has hindered structural studies. SOSIP gp140 is a soluble, proteolytically mature form of the HIV-1 envelope wherein gp120-gp41 interactions are stabilized via a disulfide bond and gp41 contains an additional trimer-stabilizing point mutation. We describe the isolation of a substantially pure preparation of SOSIP gp140 trimers derived from KNH1144, a subtype A isolate. Following initial purification, the only significant contaminant was higher-order gp140 aggregates; however, 0.05% Tween 20 quantitatively converted these aggregates into trimers. The surfactant effect was rapid, dose dependent, and similarly effective for a subtype B SOSIP gp140. Surfactant-treated SOSIP gp140 retained favorable antigenicity and formed compact trimers 12-13 nm in size as determined by electron microscopy. This report provides the first description of homogeneous, cleaved HIV-1 envelope trimers. These proteins may be useful as vaccine immunogens and for studying structure-function relationships within the HIV-1 envelope glycoproteins.