Highlights d Human cells release Argonaute 1-4 and major vault protein independently of exosomes d Annexin A1 is a specific marker of microvesicles shed from the plasma membrane d Small extracellular vesicles do not contain DNA d Active secretion of cytosolic DNA occurs through an amphisome-dependent mechanism

A complementary DNA clone encoding the large subunit of the essential mammalian pre-messenger RNA splicing component U2 snRNP auxiliary factor (U2AF65) has been isolated and expressed in vitro. It contains two functional domains: a sequence-specific RNA-binding region composed of three ribonucleoprotein-consensus sequence domains, and an arginine/serine-rich motif necessary for splicing but not for binding to pre-mRNA.

SummarySecretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.

De novo and acquired resistance, largely attributed to genetic alterations, are barriers to effective anti-EGFR therapy. We generated cetuximab-resistant cells following prolonged cetuximab exposure to cetuximab-sensitive colorectal cancer cells in three-dimensional culture. Through whole exome sequencing and transcriptional profiling, we found overexpression of lncRNA MIR100HG and two embedded miRNAs, miR-100 and miR-125b, in the absence of known genetic events linked to cetuximab resistance. MIR100HG and miR-100/125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100/125b coordinately represses five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. We describe a double-negative feedback loop between MIR100HG and GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These studies identify a clinically actionable, epigenetic cause of cetuximab resistance.

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein {PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.[Key Words: Pre-mRNA splicing; polypyrimidine tract-binding protein; PTB-associated splicing factor; cloning] Received December 9, 1992; revised version accepted January 5, 1993.The removal of intervening sequences from pre-mRNAs requires the formation of splicing complexes in an ordered, ATP-dependent pathway (for reviews, see Sharp 1987; Krainer and Maniatis 1988;Steitz et al. 1988;Smith et al. 1989a;Green 1991;Guthrie 1991). The assembly of multiple factors into these spliceosomes leads to intron excision through a two-step cleavage-ligation reaction. The development of mammalian in vitro splicing systems has allowed the identification of several cisand trans-acting components necessary for pre-mRNA splicing. Chief among the required cis-acting elements are a consensus sequence at the 5' splice site, a branchpoint sequence and adjacent polypyrimidine tract, and the 3' splice site AG dinucleotide (Mount 1982;Ohshima and Gotoh 1987). Among the best characterized of the trans-acting factors are the family of small nuclear ribonucleoprotein particles, the U snRNPs (Steitz et al. 1988). U1 and U2 snRNP initially base-pair with the 5' splice site and the branchpoint sequence, respectively, followed by the joining of U4/U6 and U5 as part of a preformed triple snRNP (Behrens and Lfihrmann 1991). Before catalysis, multiple base-pairing interactions between the pre-mRNA and small nuclear RNAs (snRNAs) Steitz 1992), multiple protein components, both snRNP and non-snRNP, are also required for pre-mRNA splicing. Each snRNP particle consists of one or two small RNAs complexed with common and unique proteins (Liihrmann 1988). Additional snRNPs are found associated with multi-snRN...