Cognate RNA Research Articles

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems.

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 Å density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

Roles of Aging, Circular RNAs, and RNA Editing in the Pathogenesis of Amyotrophic Lateral Sclerosis: Potential Biomarkers and Therapeutic Targets

Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease caused by upper and lower motor neuron death. Despite advances in our understanding of ALS pathogenesis, effective treatment for this fatal disease remains elusive. As aging is a major risk factor for ALS, age-related molecular changes may provide clues for the development of new therapeutic strategies. Dysregulation of age-dependent RNA metabolism plays a pivotal role in the pathogenesis of ALS. In addition, failure of RNA editing at the glutamine/arginine (Q/R) site of GluA2 mRNA causes excitotoxicity due to excessive Ca2+ influx through Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, which is recognized as an underlying mechanism of motor neuron death in ALS. Circular RNAs (circRNAs), a circular form of cognate RNA generated by back-splicing, are abundant in the brain and accumulate with age. Hence, they are assumed to play a role in neurodegeneration. Emerging evidence has demonstrated that age-related dysregulation of RNA editing and changes in circRNA expression are involved in ALS pathogenesis. Herein, we review the potential associations between age-dependent changes in circRNAs and RNA editing, and discuss the possibility of developing new therapies and biomarkers for ALS based on age-related changes in circRNAs and dysregulation of RNA editing.

Advantages and limitations of UV cross-linking analysis of protein-RNA interactomes in microbes.

RNA-binding proteins (RBPs) govern the lifespan of nearly all transcripts and play key roles in adaptive responses in microbes. A robust approach to examine protein-RNA interactions involves irradiating cells with UV light to form covalent adducts between RBPs and their cognate RNAs. Combined with RNA or protein purification, these procedures can provide global RBP censuses or transcriptomic maps for all target sequences of a single protein in living cells. The recent development of novel methods has quickly populated the RBP landscape in microorganisms. Here, we provide an overview of prominent UV cross-linking techniques which have been applied to investigate RNA interactomes in microbes. By assessing their advantages and caveats, this technical evaluation intends to guide the selection of appropriate methods and experimental design as well as to encourage the use of complementary UV-dependent techniques to inspect RNA-binding activity.

Decoding and Recoding of mRNA Sequences by the Ribosome.

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.

Abstract 3743: circPMS1 is a pro-metastatic circular RNA in melanoma

Abstract Deregulated gene expression is a major driver of melanoma metastasis, a process that results in the majority of melanoma-related deaths. Importantly, these alterations are not limited to protein-coding mRNAs, but also include noncoding RNAs. Despite many recent studies implicating circular RNAs (circRNAs) in cancer development, it is unknown if their deregulation contributes to melanoma metastasis. To identify circRNAs with putative roles in melanomagenesis, we performed RNA sequencing on a panel of melanoma and melanocyte cell lines. 21 differentially expressed circRNAs were validated by qPCR and Sanger sequencing of the backsplice junction. Further analyses of the ratio of the circular and cognate linear RNA transcripts and expression in a larger panel of cell lines identified circPMS1 as a circRNA that is upregulated in melanoma through enhanced backsplicing of the linear PMS1 mRNA. Using transposon-mediated delivery, we examined the effects of stable circPMS1 overexpression on melanoma cells. While proliferation and focus formation were not impacted, migration and invasion were enhanced by circPMS1. Moreover, tail vein injection of circPMS1 overexpressing melanoma cells increased lung metastasis. In addition to this transplant model, we generated the first genetically engineered mouse model of melanoma overexpressing a pro-tumorigenic circRNA. These mice are currently being analyzed. Mechanistically, we find that circularization is required for the pro-migratory effects of the circPMS1 overexpression construct. Additionally, circPMS1 harbors the canonical PMS1 start codon, a predicted ORF spanning over the backsplice junction, and a predicted IRES sequence. Indeed, inserting a flag-tag in the overexpression construct revealed circPMS1 to be translated into a truncated 25kD PMS1 protein as well as a 20kD protein likely arising from an in-frame downstream start codon. Importantly, these truncated circPMS1 proteins are endogenously expressed and increased in melanoma compared to melanocytes while the full-length PMS1 protein is evenly expressed. These findings suggest that circPMS1 promotes melanoma metastasis, at least in part, by coding for truncated circPMS1 proteins. Further work will establish the functions of the circPMS1-encoded proteins and their roles in driving melanoma progression and metastasis. Citation Format: Nicol Mecozzi, Olga Vera, Florian Karreth. circPMS1 is a pro-metastatic circular RNA in melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3743.

Functional PAM sequence for DNA interference by CRISPR-Cas I-B system of Leptospira interrogans and the role of LinCas11b encoded within lincas8b

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates into LinCas11b. A mutant variant LinCascade-Cas11b that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade-Cas11b rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system

Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.

Identification of circRNAs linked to Alzheimer's disease and related dementias.

Circular RNAs (circRNAs) exhibit selective expression in the brain and differential regulation in Alzheimer's disease (AD). To explore the role of circRNAs in AD, we investigated how circRNA expression varies between brain regions and with AD-related stress in human neuronal precursor cells (NPCs). Ribosomal RNA-depleted hippocampus RNA-sequencing data were generated. Differentially regulated circRNAs in AD and related dementias were detected using CIRCexplorer3 and limma. circRNA results were validated using quantitative real-time PCRof cDNA from the brain and NPCs. We identified 48 circRNAs that were significantly associated with AD. We observed that circRNA expression differed by dementia subtype. Using NPCs, we demonstrated that exposure to oligomeric tau elicits downregulation of circRNA similar to that observed in the AD brain. Our study shows that differential expression of circRNA can vary by dementia subtype and brain region. We also demonstrated that circRNAs can be regulated by AD-linked neuronal stress independently from their cognate linear messenger RNAs (mRNAs).

Two Alimentary Canal Proteins, Fo-GN and Fo-Cyp1, Act in Western Flower Thrips, Frankliniella occidentalis TSWV Infection.

Tomato spotted wilt virus (TSWV) is a plant virus that causes massive economic damage to high-valued crops. This virus is transmitted by specific thrips, including the western flower thrips, Frankliniella occidentalis. TSWV is acquired by the young larvae during feeding on infected host plants. TSWV infects the gut epithelium through hypothetical receptor(s) and multiplies within the cells for subsequent horizontal transmission to other plant hosts via the salivary glands during feeding. Two alimentary canal proteins, glycoprotein (Fo-GN) and cyclophilin (Fo-Cyp1), are thought to be associated with the TSWV entry into the gut epithelium of F. occidentalis. Fo-GN possesses a chitin-binding domain, and its transcript was localized on the larval gut epithelium by fluorescence in situ hybridization (FISH) analysis. Phylogenetic analysis indicated that F. occidentalis encodes six cyclophilins, in which Fo-Cyp1 is closely related to a human cyclophilin A, an immune modulator. The Fo-Cyp1 transcript was also detected in the larval gut epithelium. Expression of these two genes was suppressed by feeding their cognate RNA interference (RNAi) to young larvae. The RNAi efficiencies were confirmed by the disappearance of the target gene transcripts from the gut epithelium by FISH analyses. The RNAi treatments directed to Fo-GN or Fo-Cyp1 prevented the typical TSWV titer increase after the virus feeding, compared to control RNAi treatment. Our immunofluorescence assay using a specific antibody to TSWV documented the reduction of TSWV in the larval gut and adult salivary gland after the RNAi treatments. These results support our hypothesis that the candidate proteins Fo-GN and Fo-Cyp1 act in TSWV entry and multiplication in F. occidentalis.

Towards a Cure for HARS Disease.

Histidyl-tRNA synthetase (HARS) ligates histidine to its cognate transfer RNA (tRNAHis). Mutations in HARS cause the human genetic disorders Usher syndrome type 3B (USH3B) and Charcot-Marie-Tooth syndrome type 2W (CMT2W). Treatment for these diseases remains symptomatic, and no disease specific treatments are currently available. Mutations in HARS can lead to destabilization of the enzyme, reduced aminoacylation, and decreased histidine incorporation into the proteome. Other mutations lead to a toxic gain-of-function and mistranslation of non-cognate amino acids in response to histidine codons, which can be rescued by histidine supplementation in vitro. We discuss recent advances in characterizing HARS mutations and potential applications of amino acid and tRNA therapy for future gene and allele specific therapy.

RNA interference, an emerging component of antiviral immunity in mammals.

Antiviral RNA interference (RNAi) is an immune pathway that can, in certain conditions, protect mammalian cells against RNA viruses. It depends on the recognition and dicing of viral double-stranded RNA by a protein of the Dicer family, which leads to the production of viral small interfering RNAs (vsiRNAs) that sequence-specifically guide the degradation of cognate viral RNA. If the first line of defence against viruses relies on type-I and type-III interferons (IFN) in mammals, certain cell types such as stem cells, that are hyporesponsive for IFN, instead use antiviral RNAi via the expression of a specific antiviral Dicer. In certain conditions, antiviral RNAi can also contribute to the protection of differentiated cells. Indeed, abundant vsiRNAs are detected in infected cells and efficiently guide the degradation of viral RNA, especially in cells infected with viruses disabled for viral suppressors of RNAi (VSRs), which are virally encoded blockers of antiviral RNAi. The existence and importance of antiviral RNAi in differentiated cells has however been debated in the field, because data document mutual inhibition between IFN and antiviral RNAi. Recent developments include the engineering of a small molecule inhibitor of VSR to probe antiviral RNAi in vivo, as well as the detection of vsiRNAs inside extracellular vesicles in the serum of infected mice. It suggests that using more complex, in vivo models could allow to unravel the contribution of antiviral RNAi to immunity at the host level.

RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2

Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection1. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of ‘aromatic clamp’ residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.

Genetically programmed cell-based synthesis of non-natural peptide and depsipeptide macrocycles

The direct genetically encoded cell-based synthesis of non-natural peptide and depsipeptide macrocycles is an outstanding challenge. Here we programme the encoded synthesis of 25 diverse non-natural macrocyclic peptides, each containing two non-canonical amino acids, in Syn61Δ3-derived cells; these cells contain a synthetic Escherichia coli genome in which the annotated occurrences of two sense codons and a stop codon, and the cognate transfer RNAs (tRNAs) and release factor that normally decode these codons, have been removed. We further demonstrate that pyrrolysyl-tRNA synthetase/tRNA pairs from distinct classes can be engineered to direct the co-translational incorporation of diverse alpha hydroxy acids, with both aliphatic and aromatic side chains. We define 49 engineered mutually orthogonal pairs that recognize distinct non-canonical amino acids or alpha hydroxy acids and decode distinct codons. Finally, we combine our advances to programme Syn61Δ3-derived cells for the encoded synthesis of 12 diverse non-natural depsipeptide macrocycles, which contain two non-canonical side chains and either one or two ester bonds.

Target RNA-guided protease activity in type III-E CRISPR-Cas system.

The type III-E CRISPR-Cas systems are newly identified adaptive immune systems in prokaryotes that use a single Cas7-11 protein to specifically cleave target RNA. Cas7-11 could associate with Csx29, a putative caspase-like protein encoded by the gene frequently found in the type III-E loci, suggesting a functional linkage between the RNase and protease activities in type III-E systems. Here, we demonstrated that target RNA recognition would stimulate the proteolytic activity of Csx29, and protein Csx30 is the endogenous substrate. More interestingly, while the cognate target RNA recognition would activate Csx29, non-cognate target RNA with the complementary 3' anti-tag sequence inhibits the enzymatic activity. Csx30 could bind to the sigma factor RpoE, which may initiate the stress response after proteolytic cleavage. Combined with biochemical and structural studies, we have elucidated the mechanisms underlying the target RNA-guided proteolytic activity of Csx29. Our work will guide further developments leveraging this simple RNA targeting system for RNA and protein-related applications.

Tandemly repeated genes promote RNAi-mediated heterochromatin formation via an antisilencing factor, Epe1, in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in cooperation with an antisilencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. This "repeat-induced RNAi" depends on the copy number of repeated genes and Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

Target RNA activates the protease activity of Craspase to confer antiviral defense

In the type III-E CRISPR-Cas system, a Cas effector (gRAMP) is associated with a TPR-CHAT to form Craspase (CRISPR-guided caspase). However, both the structural features of gRAMP and the immunity mechanism remain unknown for this system. Here, we report structures of gRAMP-crRNA and gRAMP:cRNA:target RNA as well as structures of Craspase and Craspase complexed with cognate target RNA (CTR) or non-cognate target RNA (NTR). Importantly, the 3' anti-tag region of NTR and CTR binds at two distinct channels in Craspase, and CTR with a non-complementary 3' anti-tag induces a marked conformational change of the TPR-CHAT, which allosterically activates its protease activity to cleave an ancillary protein Csx30. This cleavage then triggers an abortive infection as the antiviral strategy of the type III-E system. Together, our study provides crucial insights into both the catalytic mechanism of the gRAMP and the immunity mechanism of the type III-E system.

Association of ScV-LA Virus with Host Protein Metabolism Determined by Proteomics Analysis and Cognate RNA Sequencing.

Saccharomyces yeasts are highly dispersed in the environment and microbiota of higher organisms. The yeast killing phenotype, encoded by the viral system, was discovered to be a significant property for host survival. Minor alterations in transcription patterns underpin the reciprocal relationship between LA and M viruses and their hosts, suggesting the fine-tuning of the transcriptional landscape. To uncover the principal targets of both viruses, we performed proteomics analysis of virus-enriched subsets of host proteins in virus type-specific manner. The essential pathways of protein metabolism-from biosynthesis and folding to degradation-were found substantially enriched in virus-linked subsets. The fractionation of viruses allowed separation of virus-linked host RNAs, investigated by high-content RNA sequencing. Ribosomal RNA was found to be inherently associated with LA-lus virus, along with other RNAs essential for ribosome biogenesis. This study provides a unique portrayal of yeast virions through the characterization of the associated proteome and cognate RNAs, and offers a background for understanding ScV-LA viral infection persistency.

Promiscuity of response regulators for thioredoxin steers bacterial virulence

The exquisite specificity between a sensor kinase and its cognate response regulator ensures faithful partner selectivity within two-component pairs concurrently firing in a single bacterium, minimizing crosstalk with other members of this conserved family of paralogous proteins. We show that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using the OmpR protein, we identify residues in the flexible linker and the C-terminal β-hairpin that enable associations of this archetypical response regulator with thioredoxin, but are dispensable for interactions of this transcription factor to its cognate sensor kinase EnvZ, DNA or RNA polymerase. Here we show that the promiscuous interactions of response regulators with thioredoxin foster the flow of information through otherwise highly dedicated two-component signaling systems, thereby enabling both the transcription of Salmonella pathogenicity island-2 genes as well as growth of this intracellular bacterium in macrophages and mice.

Changes of Protein Expression after CRISPR/Cas9 Knockout of miRNA-142 in Cell Lines Derived from Diffuse Large B-Cell Lymphoma.

Simple SummaryThe gene of the human tumor suppressive microRNA-142 (miR-142) carries mutations in about 20% of cases of diffuse large B-cell lymphoma (DLBCL). Because microRNAs post-transcriptionally regulate the protein expression of their cognate messenger RNA (mRNAs) targets, we determined the effect of miR-142 knockout on protein expression in two cell lines derived from DLBCL. We found a significant up-regulation of 52 proteins but also a down-regulation of 41 proteins upon miR-142 deletion. Knockout of a miRNA may be used to identify novel targets, and seed-sequence mutants of a miRNA unable to bind to their targets can be used to confirm potential novel targets. With this approach, we identify AKT1S1, CCNB1, LIMA1 and TFRC as novel targets of miR-142. As miR-142 is highly present in the miRNA processing RISC complexes, the deletion of this miRNA might result in its replacement by other miRNAs, thus introducing an additional layer of complexity regarding gene regulation.Background: As microRNA-142 (miR-142) is the only human microRNA gene where mutations have consistently been found in about 20% of all cases of diffuse large B-cell lymphoma (DLBCL), we wanted to determine the impact of miR-142 inactivation on protein expression of DLBCL cell lines. Methods: miR-142 was deleted by CRISPR/Cas9 knockout in cell lines from DLBCL. Results: By proteome analyses, miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. Conclusions: Seed-sequence mutants of miR-142 confirmed potential targets and novel targets of miRNAs can be identified in miRNA knockout cell lines. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when miRNAs highly present in RISC complexes are replaced by other miRNAs, primary effects on gene expression may be covered by secondary layers of regulation.