The TGF-ß-inducible miR-23a cluster attenuates IFN-γ levels and antigen-specific cytotoxicity in human CD8⁺ T cells.

Cytokine secretion and degranulation represent key components of CD8(+) T-cell cytotoxicity. While transcriptional blockade of IFN-γ and inhibition of degranulation by TGF-ß are well established, we wondered whether TGF-ß could also induce immune-regulatory miRNAs in human CD8(+) T cells. We used miRNA microarrays and high-throughput sequencing in combination with qRT-PCR and found that TGF-ß promotes expression of the miR-23a cluster in human CD8(+) T cells. Likewise, TGF-ß up-regulated expression of the cluster in CD8(+) T cells from wild-type mice, but not in cells from mice with tissue-specific expression of a dominant-negative TGF-ß type II receptor. Reporter gene assays including site mutations confirmed that miR-23a specifically targets the 3'UTR of CD107a/LAMP1 mRNA, whereas the further miRNAs expressed in this cluster-namely, miR-27a and -24-target the 3'UTR of IFN-γ mRNA. Upon modulation of the miR-23a cluster by the respective miRNA antagomirs and mimics, we observed significant changes in IFN-γ expression, but only slight effects on CD107a/LAMP1 expression. Still, overexpression of the cluster attenuated the cytotoxic activity of antigen-specific CD8(+) T cells. These functional data thus reveal that the miR-23a cluster not only is induced by TGF-ß, but also exerts a suppressive effect on CD8(+) T-cell effector functions, even in the absence of TGF-ß signaling.

CAMTA1 is a novel tumour suppressor regulated by miR-9/9* in glioblastoma stem cells.

Cancer stem cells or cancer initiating cells are believed to contribute to cancer recurrence after therapy. MicroRNAs (miRNAs) are short RNA molecules with fundamental roles in gene regulation. The role of miRNAs in cancer stem cells is only poorly understood. Here, we report miRNA expression profiles of glioblastoma stem cell-containing CD133(+) cell populations. We find that miR-9, miR-9(*) (referred to as miR-9/9(*)), miR-17 and miR-106b are highly abundant in CD133(+) cells. Furthermore, inhibition of miR-9/9(*) or miR-17 leads to reduced neurosphere formation and stimulates cell differentiation. Calmodulin-binding transcription activator 1 (CAMTA1) is a putative transcription factor, which induces the expression of the anti-proliferative cardiac hormone natriuretic peptide A (NPPA). We identify CAMTA1 as an miR-9/9(*) and miR-17 target. CAMTA1 expression leads to reduced neurosphere formation and tumour growth in nude mice, suggesting that CAMTA1 can function as tumour suppressor. Consistently, CAMTA1 and NPPA expression correlate with patient survival. Our findings could provide a basis for novel strategies of glioblastoma therapy.

Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance.

Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449-resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

A human snoRNA with microRNA-like functions.

Small noncoding RNAs function in concert with Argonaute (Ago) proteins to regulate gene expression at the level of transcription, mRNA stability, or translation. Ago proteins bind small RNAs and form the core of silencing complexes. Here, we report the analysis of small RNAs associated with human Ago1 and Ago2 revealed by immunoprecipitation and deep sequencing. Among the reads, we find small RNAs originating from the small nucleolar RNA (snoRNA) ACA45. Moreover, processing of ACA45 requires Dicer activity but is independent of Drosha/DGCR8. Using bioinformatic prediction algorithms and luciferase reporter assays, we uncover the mediator subunit CDC2L6 as one potential mRNA target of ACA45 small RNAs, suggesting a role for ACA45-processing products in posttranscriptional gene silencing. We further identify a number of human snoRNAs with microRNA (miRNA)-like processing signatures. We have, therefore, identified a class of small RNAs in human cells that originate from snoRNAs and can function like miRNAs.

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

A multifunctional human Argonaute2-specific monoclonal antibody.

Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.

Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells.

Members of the Argonaute (Ago) protein family associate with small RNAs and have important roles in RNA silencing. Here, we analysed Ago1- and Ago2-containing protein complexes in human cells. Separation of Ago-associated messenger ribonucleoproteins (mRNPs) showed that Ago1 and Ago2 reside in three complexes with distinct Dicer and RNA-induced silencing complex activities. A comprehensive proteomic analysis of Ago-containing mRNPs identified a large number of proteins involved in RNA metabolism. By using co-immunoprecipitation experiments followed by RNase treatment, we biochemically mapped interactions within Ago mRNPs. Using reporter assays and knockdown experiments, we showed that the putative RNA-binding protein RBM4 is required for microRNA-guided gene regulation.