The Roles of Individual Mammalian Argonautes in RNA Interference In Vivo

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins.Alnylam Pharmaceuticals (Firm

Caffeine Prevents Transcription Inhibition and P-TEFb/7SK Dissociation Following UV-Induced DNA Damage

Background: The mechanisms by which DNA damage triggers suppression of transcription of a large number of genes are poorly understood. DNA damage rapidly induces a release of the positive transcription elongation factor b (P-TEFb) from the large inactive multisubunit 7SK snRNP complex. P-TEFb is required for transcription of most class II genes through stimulation of RNA polymerase II elongation and cotranscriptional pre-mRNA processing. Methodology/Principal Findings: We show here that caffeine prevents UV-induced dissociation of P-TEFb as well as transcription inhibition. The caffeine-effect does not involve PI3-kinase-related protein kinases, because inhibition of phosphatidylinositol 3-kinase family members (ATM, ATR and DNA-PK) neither prevents P-TEFb dissociation nor transcription inhibition. Finally, caffeine prevention of transcription inhibition is independent from DNA damage. Conclusion/Significance: Pharmacological prevention of P-TEFb/7SK snRNP dissociation and transcription inhibitio

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)[superscript +] macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE[superscript −/−] mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques

Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered

Effect of individual Argonautes in vivo.

<p>(A) C57BL/6 mice were i.v.-injected with LNP-formulated siRNA targeting Ago2–3′UTR or with control Luciferase siRNA at 0.5 mg/kg. Animals were sacrificed at indicated time points and Ago2 mRNA was quantified by bDNA assay (mean ± s.d., n = 5). (B) bDNA measurement of mRNA knockdown of Rab5c (by siRNA targeting 3′UTR-near at 0.5 mg/kg) and FVII (by siRNA targeting CDS at 0.2 mg/kg) in wild-type mice (mean ± s.d., n = 4–5) following 4 days of Ago2 knockdown (by siRNA targeting 3′UTR at 0.5 mg/kg). (C) Animals were i.v.-injected with Ago1, Ago2, or Luc siRNA on day 1, Ago1 treated animals were further i.v.-injected with Ago2 on day 2; and Rab5c-3′UTR-near siRNA was introduced on day 5 (where indicated; each siRNA was dosed at 0.5 mg/kg). All animals were sacrificed on day 6. Liver mRNA levels for Ago1, Ago2, and Rab5c were measured by bDNA assay (mean ± s.d., n = 4–5). (D) Mice were i.v.-injected with LNP containing 0.5 mg/kg siRNA against each of Ago1 and Ago2 (or 1 mg/kg of Luciferase control siRNA) twice at week long intervals, at the end of the second week mice were i.v.-injected with Fads1–3′UTR or Luciferase control siRNAs at 0.4 mg/kg. Mice were sacrificed for liver RNA extraction the day after the last injection (mean ± s.d., n = 3).</p

siRNAs targeting CDS and 3′UTR differ in their Ago2 dependence <i>in vivo</i>.

<p>Data points are levels of mRNA measured by qRT-PCR in the livers (harvested 24 hours post-injection) of mice i.v.-injected with LNP-formulated siRNAs, as described in Materials and Methods, expressed as a percentage of mRNA levels in control siRNA-treated animals. (A) mRNA expression of Argonautes and Dicer1 in Ago2^fl/fl and Ago2^−/− mouse liver (mean ± s.d., n = 4). (B) Levels of Fads1 mRNA in livers of Ago2^−/− mice and Ago2^fl/fl mice (mean ± s.d., n = 2–3) treated with siRNAs targeting Fads1 CDS or 3′UTR at 0.5 mg/kg. (C) Rab5c mRNA levels after treatment with siRNAs targeting its CDS or 3′UTR-far at 1 mg/kg (mean ± s.d., n = 2–3). (D) FVII mRNA in the liver of animals treated with siRNAs (mean ± s.d., n = 4–5, doses: 0.8 mg/kg for siRNA targeting CDS and 0.4 mg/kg for siRNA targeting 3′UTR) and FVII protein in the serum quantified by chromogenic assay. (E) Activity of siRNAs targeting CDS and 3′UTR in the absence of Ago1, 3, or 4. Legend in the bottom left corner of the graph also indicates the line in the X-axis describing the treatment type by gene (mean ± s.d., n = 3). Mice knockout for different individual Argonaute genes and C57BL/6 control animals were i.v.-injected with one of two combinations of LNP-formulated siRNAs targeting Fads1 and Rab5c (CDS-targeting for one gene, at 0.8 mg/kg and 3′UTR-targeting for the other gene, at 0.4 mg/kg) or with control Luciferase siRNA at 1.2 mg/kg.</p

Differential Ago2 dependence of siRNAs targeting coding sequence (CDS) and 3′-untranslated region (3′UTR) of mRNA <i>in vitro</i>.

<p>(A) Mouse embryonic fibroblast (MEF) cells, Ago2^fl/fl and Ago2^−/− (wild-type and Ago2 knockout) were transfected with 10 nM of siRNAs targeting CDS or 3′UTR (16 or 28 siRNAs, respectively, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101749#pone.0101749.s004" target="_blank">Table S1</a>) of mRNAs of four genes: Fads1, Fads2, Rab5a, and Rab5c. Levels of expression of target-gene mRNA in Ago2^fl/fl MEF cells and Ago2^−/− MEF cells measured by branched DNA (bDNA) assay 24 h post-transfection were plotted as percentage of relative target mRNA compared to Luciferase siRNA transfected controls. (B, C) Ago2^−/− MEF cells (square) or Ago2^fl/fl MEF cells (circle) were transfected with siRNAs targeting CDS or 3′UTR of Fads1 (B) or Rab5c (C) mRNA (relative positions of target sites within mRNA are shown). Transfection and assay as described in A, but dilution series with the maximum dose of 40 nM were done. Combined results of two independent transfections are shown. (D) Ago2^fl/fl MEF cells were transfected with 10 nM siRNA targeting 3′UTR of Ago2 (Eif2c2) mRNA or Luciferase, followed by transfection with two combinations of siRNAs targeting Fads1 and Rab5c (CDS-targeting for one gene and 3′UTR-targeting for the other gene, 10 nM each, or 20 nM of Luciferase siRNA control) 48 hours later. Levels of target-genes and Ago2 mRNA expression measured by bDNA assay 24 h after second transfection were plotted as percentage of relative target mRNA compared to Luciferase siRNA-transfected control (mean ± s.d., n = 3). Legend in the bottom left corner of the graph also indicates the line in the X-axis describing the treatment type (none, CDS, 3′UTR) by target gene.</p

Cleavage independent siRNA-mediated degradation of mRNA in the absence of Ago2.

<p>Ago2^fl/fl MEF cells and Ago2^−/− MEF cells were transfected with 10 nM of siRNAs targeting Luciferase, or CDS, or 3′UTR of the Rab5c mRNA. bDNA assay (A) and 5′RACE (B) were performed on total RNA isolated from cells 3 h post-transfection. (A) Data is presented as mean ± s.d. for two technical replicates of bDNA measurement. (B) 5′RACE nested PCR detection of cleavage product. Numbers of clones bearing expected inserts are indicated below corresponding lanes of the gel (expected PCR product length is 145 bp; expected adapter/Rab5c junction sequence is shown below; 16 clones were sequenced for each PCR reaction).</p