Evidence for the biogenesis of more than 1,000 novel human microRNAs

Background: MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome. Results: Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain. Conclusions: In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.This project was funded by the Spanish Plan Nacional SAF2008-00357 (NOVADIS); the Generalitat de Catalunya AGAUR 2009 SGR-1502; the Instituto de Salud Carlos III (FIS/FEDER PI11/00733); National Institutes of Health (R00HG004515 to KCC) and the European Commission 7th Framework Program, Projects N. 03790 (SIROCCO), N. 282510 (BLUEPRINT), N. 261123 (GEUVADIS) and N. 262055 (ESGI). MRF is supported by EMBO Long-Term fellowship ALTF 225–2011; EL is supported by the ICGC CLL-Genome project funded by the Spanish Ministry of Economy and Competiveness through the Instituto de Salud Carlos III. AJSH is a Marie Curie postdoctoral fellow supported by the European Commission 7th Framework Program under grant agreement N. 330133. MB-C is a Sara Borrell postdoctoral fellow supported by the Spanish Ministry of Economy and Competiveness. GK was supported by the Wellcome Trust Grant 097383 and by the MRC. EM–H is a PhD student from LaCaix

A Pathogenic Mechanism in Huntington's Disease Involves Small CAG-Repeated RNAs with Neurotoxic Activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches

Insight into disease processes of fragil X premutation carriers associated pathologies : expression-profile characterization and identification of a novel pathogenic mechanisms

Male premutation carriers (PM) presenting between 55-200 CGG repeats in the Fragile X-associated (FMR1) gene are at risk to develop Fragile X Tremor/Ataxia Syndrome (FXTAS), and females to undergo Premature Ovarian Failure (POF1). These pathologies are caused by toxic gain of function of the premutated FMR1 mRNA. Functional alterations of several gene expression regulators provide a detrimental mechanism underlying FMR1 PM–associated pathologies. In this thesis, we have characterized the transcriptome alterations associated to FMR1 premutation and further characterized the relevance of the biogenesis and activity of a small RNAs formed by repeated CGG (sCGG) in neuronal dysfunction linked to FMR1-PM. In blood of FMR1 premutation carriers (fXPCs) we have detected a strong deregulation of genes enriched in FXTAS-relevant biological pathways. We have also identified a deregulated gene (EAP1) that may underlie POF1 in female fXPCs. In addition, we found increased levels of sCGG in different models of FMR1-PM and further demonstrated the neurotoxic activity of sCGG through a mechanism dependent on RNA induced silencing machinery. We propose that the activity of sCGG may contribute to transcriptome perturbations with downstream pathogenic consequences. Overall, we provide mechanistic insight into the disease process and further suggest targets for FXTAS diagnosis to the myriad of phenotypes associated with FXPC.Homes portadors de la premutació (PM) en el gen associat Fràgil X (FMR1), presenten entre 55-200 repeticions de CGG, estan en risc de desenvolupar el síndrome de tremolor/atàxia associat al X fràgil (FXTAS), i les dones fallida ovàrica precoç (POF1). Aquestes malalties són causades per la funció tòxica de l'ARN missatger. Alteracions funcional de diversos reguladors de l'expressió gènica s'ha proposat com a causa subjacent a aquests trastorns. En aquesta tesi, s'han caracteritzat les alteracions associades al transcriptome de la premutació en l’FMR1 i analitzat la rellevància de la biogènesi i l'activitat d'un petit ARN format per CGG repetits (sCGG) en la disfunció neuronal relacionada amb la PM del FMR1. En sang de portadors de la premutació en l’FMR1 (fXPCs) s'ha detectat una forta desregulació de gens enriquit en vies biològiques rellevants en FXTAS. També hem identificat un gen desregulat (EAP1) que pot ser la base POF1 en dones fXPCs. A més, hem trobat un augment en els nivells de sCGG en diferents models de FMR1-PM i demostrem la seva activitat neurotòxica a través d'un mecanisme dependent en la maquinària de silenciament gènic. Proposem que l'activitat de sCGG pot contribuir a causar pertorbacions en el transcriptoma i desencadenar conseqüències patògenes.En general, oferim un nou enfoc en procés de la malaltia i un diagnòstic més exacte per la gran varietat de fenotips associats amb fXPCs

A pathogenic mechanism in Huntington"s disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approache

A pathogenic mechanism in Huntington"s disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approache

sCAG neurotoxic effect is dependent on Dicer and Ago proteins.

<p>A. Dicer knockdown inhibits the generation of sCAGs produced by the expression of 80*CAG HTT-e1. sCAG levels were normalized to RNU66 levels. GFP blots indicate the expression of the HTT-constructs (n = 3; interaction p-value = 0.000138; F = 46.220). B. In the same experiments, cell viability and caspase 9 cleavage analysis show that Dicer depletion mitigates cell death induced by expanded <i>HTT</i> (n = 5; interaction p-value = 0.000135; F = 18.263). C. Ago2 depletion mitigates the toxicity of sRNA obtained from mutant <i>HTT</i> expressing cells (n = 3; interaction p-value = 0.011; F = 10.821). D. sCAG efficiently associate to Ago2 <i>in vivo</i>. <i>HTT</i>-expressing constructs were transfected on cells stably expressing Flag-Ago2. Flag IP demonstrate that sCAG binds to Ago2 complex. No significant binding was detected in control IP experiments (α-V5). The plot shows the mean ratio of sCAG levels in FLAG IP <i>vs.</i> control V5 IP (n = 3; *p<0.05). E. The expression of Flag-Ago2 in cells depleted for endogenous Ago2 partially, but significantly, rescued CAG-expanded HTT toxic effect (n = 3; *p<0,05). Values represent the mean of the ratio expanded-HTT sRNA toxicity vs non-expanded-HTT sRNA toxicity ± SEM in each experimental condition. Toxicity levels are referred to the control cells lacking HTT expression. In A. B. and C., values represent the mean fold change with respect to the control, non-transfected cells ± SEM. Cells were processed 24 hours after double transfection in all the experiments.</p

Expanded HTT generates CAG-repeated sRNAs with toxic activity.

<p>A. sRNA fraction (<100 nt) were isolated from cells expressing HTT-e1 constructs and equal amounts of each pool were transfected. Both 80*CAG-RNA and 80*CAG-PROT- derived sRNA pools induced death of differentiated SH-SY5Y cells (n = 5; *p<0.05,**p<0.01). 80*CAA-PROT-derived sRNA pools didn't affect SH-SY5Y cell viability. B. The expression of CAG-expanded HTT leads to an increase in CAG-repeated sRNAs of ∼21-nt (sCAG). sCAG levels were quantified using RNU66 as the reference sRNA, and normalized with respect to GFP expression, which indicates the percentage on transfected living cells 24 hours after transfection (n = 4; **p<0.01). C. HTT sRNA toxicity correlates with the length of the CAG expansion, distinguishing pathogenic and non-pathogenic number of CAG repeats (n = 4; * p<0.05, **p<0.01 ***p<0.001. D. HTT sRNA toxicity correlates with the generation of sCAG species (n = 4; **p<0.01). E. Anti-(CAG)₇ sRNA (anti-sCAG) prevents cell damage caused by mutant-<i>HTT</i>-derived sRNA pool. Control sRNA inhibitors did not mitigate sRNA HTT toxicity (n = 4; *p<0.05, **p<0.01, **p<0.001, determinations were performed in quintuplicates). Values represent mean of the ratio expanded-HTT sRNA toxicity vs non-expanded-HTT sRNA toxicity ± SEM. In A. B. C. and D. values represent the mean fold change with respect to the control non-transfected cells ± SEM and are referred to the control cells lacking HTT expression. In all experiments, cells were processed 24 hours after transfection in all the experiments.</p

Cytotoxic sCAGs are increased in brain regions of HD.

<p>A. sCAG levels are increased in affected brain areas from R6/2 HD mouse model compared to control mice. sCAG were quantified by qRT-PCR using RNU6B as the reference sRNA; HC, hippocampus; STR, striatum cortex; CX, cortex; and CB, cerebellum. Values represent mean fold change with respect the control samples ± SEM (n = 3; *p<0.05 ***p<0.001). B. Increased expression of sCAG in HD human brain samples compared to control subjects. CA, caudate; and FC, frontal cortex. RNU66 sRNA was used as reference sRNA. Values represent mean fold change with respect to the control samples ± SEM (n = 3; *p<0.05 ***p<0.001). C. HD-derived sRNA pools induce neuronal toxicity. sRNA pools were isolated from control and HD human brain samples and delivered to differentiated SH-SY5Y cells; cell death was determined 24 hours later. The use of anti-sCAG dramatically reduced the cytotoxic effect. Control sRNA inhibitors (scrambled anti-sRNA) were used as a negative control. Values represent mean of the ratio (HD sRNA toxicity/Control sRNA toxicity) for each condition ± SEM (experiments were performed in quintuplicates, n = 6; *p<0.05). Pools from four control individuals and four patients with HD were used.</p

sCAGs induce post-transcriptional gene silencing in genes with CTG regions.

<p>A. Hela cells were cotransfected with firefly luciferase expressing vectors containing the indicated nucleotide sequences in its 3′-UTR, the specific HTT-e1 expressing vectors or the (CAG)7 siRNA and Renilla luciferase plasmid to normalize data. Assays were performed 24 hours after transfection. Data were first normalized to the 100% of luminescence obtained with the control luciferase vector, lacking 3′UTR inserts (n = 3; *p<0.05, p**p<0,01). B,C. Levels of ADORA2A and MEIS2 transcripts in SH-SY5Y cells transfected with normal and expanded HTT vectors. MRIP was used as endogenous control. qRT-PCR was performed in cells fixed 24 hours after transfection. (n = 3; *p<0.05). Values represent the mean fold change with respect to the control, non-transfected cells ± SEM. D. Western blot showing reduced MEIS2 protein levels in differentiated SH-SY5Y expressing expanded <i>HTT</i> RNA 24 hours after transfection. The graph shows the densitometry determination of MEIS2 levels <i>vs</i> β-Actin. Results represent the mean arbitrary optical density change normalized to the mean value obtained in non-transfected cells (n = 4; *p<0.05).</p

sCAG toxicity is variable in different human cells, preferentially affecting neuronal viability.

<p>A. Exogenous administration of (CAG)₇ siRNAs interfere with cell viability depending on the cell type (n = 3, p<0.0083). HMEC, HPDE and UROTSA cell lines were used as a source for breast, pancreatic and bladder primary human cells. Differentiated SH-SY5Y cells were used as a post-mitotic neuronal cell model (n = 3; one-way ANOVA *p<0.05 ***p<0.001; F = 15.203). B. The toxic effect of (CAG)7 siRNA is dependent on the type of differentiation. SHSY5Y cells were subjected to several neuronal differentiation protocols, and CAG)₇ or scrambled sequences (control siRNA) were administered in each situation. SH-SY5Y sensitivity to (CAG)7 significantly differs in each differentiation condition (*), excepting for TPA standard differentiation condition (#), whose effect wasn't significantly different from the effect observed under TPA long exposure conditions (One-way ANOVA; F = 63.926). (n = 3, p<0.0083). MTT assays were performed 48 hours after transfection. Graphs show relative cell survival indicated as the ratio between cell viability in cells transfected with controls siRNA <i>vs</i> cell viability in cells transfected with (CAG)7. Values indicate the mean ratio ± SEM of three independent experiments.</p