mRNA In Mammalian Cells Research Articles

Sumoylation is Required for the Cytoplasmic Accumulation of a Subset of mRNAs.

In order to discover novel proteins that promote the nuclear export of newly synthesized mRNAs in mammalian cells, we carried out a limited RNAi screen for proteins required for the proper cytoplasmic distribution of a model intronless mRNA. From this screen we obtained two hits, Ubc9 (SUMO-conjugating E2 enzyme) and GANP (germinal center-associated nuclear protein). Depletion of Ubc9 inhibited the proper cytoplasmic distribution of certain overexpressed intronless mRNAs, while depletion of GANP affected all tested mRNAs. Depletion of Sae1, which is also required for sumoylation, partially inhibited the cytoplasmic distribution of our model mRNA. Interestingly, the block in cytoplasmic accumulation in Ubc9-depleted cells could be overcome if an intron was incorporated into the mRNA. Surprisingly, Ubc9-depleted cells had normal nuclear export of newly synthesized intronless mRNAs, indicating that the observed accumulation of the model mRNA in the nuclei of transfected cells was likely due to some more general perturbation. Indeed, depletion of Ubc9, coupled with the overexpression of the intronless mRNAs, caused the redistribution of the nuclear speckle protein SC35 to cytoplasmic foci. Our results suggest that sumoylation may play a role in the proper assembly of mRNPs and/or the distribution of key RNA binding proteins, and may thus contribute to general protein expression patterns.

Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems.

Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.

Highly Complementary Target RNAs Promote Release of Guide RNAs from Human Argonaute2

Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This "unloading" activity can be enhanced by mismatches between the target and the guide 5' end and attenuated by mismatches to the guide 3' end. The introduction of 3' mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3' ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells.

Abstract 4097: Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell.

Abstract The mammalian target rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of mRNA in mammalian cells. Torin2 is a potent, selective and orally available mTOR inhibitor for treatment of cancer. Dysregulation of mTOR signaling has been found in many cancers, however its role Papillary Thyroid Cancer (PTC) has not been explained. Therefore in this study, we investigated the role of mTOR and its associated molecules in PTC cell lines using MTT, flow cytometry and DNA fragmentation assays and western blotting. Our data showed that Torin2 caused a dose dependent growth inhibition in all cell lines studied. Cell cycle assay showed that the sub-G1 population in TPC-1 cell increased from 12.44% in untreated control sample to 31.50% after 200nM of Torin2 treatment and 44.08% after 400nM of Torin2 treatment for 48 hours. Similarly BCPAP cells increased from 8.47% in untreated control sample to 33.54% after 200nM of Torin2 treatment and 41.16% after 400nM of Torin2 treatment for 48 hours. Further confirmation using FITC-conjugated Annexin V and PI assay showed that apoptotic cell increased from 8.89% in untreated cells to 58.54% and 73.54% after 200nM and 400nM treatment in TPC-1 cells, and from 13.24% in control to 46.74% and 70.64% in BCPAP cells, indicating a dose dependent apoptosis induced in both cell lines. Treatment of PTC cell lines with Torin2 inactivated downstream targets of mTOR; p70S6 and 4E-BP1. Torin2 treatment of PTC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspase-9, and 3, and cleavage of PARP. Furthermore Torin2 positively regulated autophagy by disrupting the Beclin-1 complex, releasing Beclin-1 to induce autophagy. Our results suggest mTOR and its associated signaling pathway play a critical role in PTC tumorigenesis, therefore targeting of mTOR expression may be a viable therapeutic intervention for treatment of these cancers. Citation Format: Maqbool Ahmed, Azhar R. Hussain, Fouad Al Dayel, Shahab Uddin, Khawla S. Al-Kuraya. Targeting of mTOR mediated signaling pathways in papillary thyroid cancer cell. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2013-4097

Experimental characterization of the human non-sequence-specific nucleic acid interactome

BackgroundThe interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.ResultsWe designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function.ConclusionsThe dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

The PARN Deadenylase Targets a Discrete Set of mRNAs for Decay and Regulates Cell Motility in Mouse Myoblasts

PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3′ UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre–mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement.

Selenocysteine Insertion Sequence Binding Protein 2L Is Implicated as a Novel Post-Transcriptional Regulator of Selenoprotein Expression

The amino acid selenocysteine (Sec) is encoded by UGA codons. Recoding of UGA from stop to Sec requires a Sec insertion sequence (SECIS) element in the 3′ UTR of selenoprotein mRNAs. SECIS binding protein 2 (SBP2) binds the SECIS element and is essential for Sec incorporation into the nascent peptide. SBP2-like (SBP2L) is a paralogue of SBP2 in vertebrates and is the only SECIS binding protein in some invertebrates where it likely directs Sec incorporation. However, vertebrate SBP2L does not promote Sec incorporation in in vitro assays. Here we present a comparative analysis of SBP2 and SBP2L SECIS binding properties and demonstrate that its inability to promote Sec incorporation is not due to lower SECIS affinity but likely due to lack of a SECIS dependent domain association that is found in SBP2. Interestingly, however, we find that an invertebrate version of SBP2L is fully competent for Sec incorporation in vitro. Additionally, we present the first evidence that SBP2L interacts with selenoprotein mRNAs in mammalian cells, thereby implying a role in selenoprotein expression.

Two isoforms of the mRNA binding protein IGF2BP2 are generated by alternative translational initiation.

IGF2BP2 is a member of a family of mRNA binding proteins that, collectively, have been shown to bind to several different mRNAs in mammalian cells, including one of the mRNAs encoding insulin-like growth factor-2. Polymorphisms in the Igf2bp2 gene are associated with risk of developing type 2 diabetes, but detailed functional characterisation of IGF2BP2 protein is lacking. By immunoblotting with C-terminally reactive antibodies we identified a novel IGF2BP2 isoform with a molecular weight of 58 kDa in both human and rodents, that is expressed at somewhat lower levels than the full-length 65 kDa protein. We demonstrated by mutagenesis that this isoform is generated by alternative translation initiation at the internal Met69. It lacks a conserved N-terminal RNA Recognition Motif (RRM) and would be predicted to differ functionally from the canonical full length isoform. We further investigated IGF2BP2 mRNA transcripts by amplification of cDNA using 5′-RACE. We identified multiple transcription start sites of the human, mouse and rat Igf2bp2 genes in a highly conserved region only 50–90 nts upstream of the major translation start site, ruling out the existence of N-terminally extended isoforms. We conclude that structural heterogeneity of IGF2BP2 protein should be taken into account when considering cellular function.

Regulation of cytoplasmic mRNA decay

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means of modulating gene expression and thereby protein production. Up until recently, studies largely focused on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay and the ribonucleases that catalyse decay. Now, current studies have begun to elucidate how the decay process is regulated. This Review examines our current understanding of how mammalian cell mRNA decay is controlled by different signalling pathways and lays out a framework for future research.

A novel function of Tis11b/BRF1 as a regulator ofDll4mRNA 3′-end processing

Tis11b/BRF1 belongs to the tristetraprolin family, the members of which are involved in AU-rich-dependent regulation of mRNA stability/degradation. Mouse inactivation of the Tis11b gene has revealed disorganization of the vascular network and up-regulation of the proangiogenic factor VEGF. However, the VEGF deregulation alone cannot explain the phenotype of Tis11b knockouts. Therefore we investigated the role of Tis11b in expression of Dll4, another angiogenic gene for which haploinsufficiency is lethal. In this paper, we show that Tis11b silencing in endothelial cells leads to up-regulation of Dll4 protein and mRNA expressions, indicating that Dll4 is a physiological target of Tis11b. Tis11b protein binds to endogenous Dll4 mRNA, and represses mRNA expression without affecting its stability. In the Dll4 mRNA 3' untranslated region, we identified one particular AUUUA motif embedded in a weak noncanonical polyadenylation (poly(A)) signal as the major Tis11b-binding site. Moreover, we observed that inhibition of Tis11b expression changes the ratio between mRNAs that are cleaved or read through at the poly(A) signal position, suggesting that Tis11b can interfere with mRNA cleavage and poly(A) efficiency. Last, we report that this Tis11b-mediated mechanism is used by endothelial cells under hypoxia for controlling Dll4 mRNA levels. This work constitutes the first description of a new function for Tis11b in mammalian cell mRNA 3'-end maturation.

Inducible and reversible breaching of the blood brain barrier by RNAi

Sequence-specific knockdown of gene expression is a goal that has been long sought by both basic and clinical investigators. In this regard, the discovery of RNA interference (RNAi) in Caenorhabditis elegans was immediately recognized as a potential breakthrough for studying gene function (Fire et al, 1998). These findings demonstrated that double-stranded (ds)RNAs are triggers for sequence-specific, post-transcriptional gene silencing via targeted degradation of messenger RNAs harbouring a complementary sequence to one of the two strands. Initially, it was thought that such post-transcriptional regulation of gene expression could not be achieved in mammalian systems due to the strong induction of interferon by dsRNAs. This potential restriction was short lived with the demonstration that endonuclease processed dsRNAs of 21–25 nucleotides in length, designated small interfering RNAs (siRNAs), were able to elicit sequence-specific degradation of mRNAs in mammalian cells without triggering interferon responses (Elbashir et al, 2001). These findings provided a huge impetus to develop RNAi as a therapeutic modality. The dream to selectively block the expression of deleterious proteins and treat formerly non-drugable diseases led to the rapid establishment of new biotech companies and branches of major pharmaceutical companies devoted to RNAi therapeutics.

Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

Site-specific cleavage of CD59 mRNA by endoplasmic reticulum-localized ribonuclease, IRE1

IRE1, an ER-localized transmembrane-RNase, plays a central role in ER stress response. Upon ER stress, IRE1 induces various adaptive genes through the processing of mRNA encoding the transcription factor XBP1. Moreover, it was recently reported that in fly IRE1 attenuates the expression of several genes by cleaving mRNAs, but it has been unclear whether such a mechanism also exists in mammal. In this study, we searched for IRE1α-cleaved mRNAs in mammalian cells and identified human CD59 (complement defense 59) mRNA as a novel cleavage target. In addition, the expression of CD59 was significantly attenuated by overexpression of IRE1α or ER stress. These results suggest that IRE1α-mediated mRNA cleavage functions even in mammals as a common system to regulate gene expression.

Liposome-mediated RNA transfection should be used with caution

Liposome-mediated RNA transfection appears to present a number of advantages for studying the metabolism of reporter mRNAs in mammalian cells. This method is also widely used to transfect siRNAs. Here we describe results indicating that reporter mRNAs introduced into HeLa cells by liposomes do not present the expected behaviors. Namely, the stability of reporter mRNAs was independent of the presence or absence of an AUUUA instability element, a poly(A) tail, or even a 5' methylated cap. Confocal microscopy showed that fluorescent RNAs introduced by liposome-mediated transfection were present in discrete particles. These observations imply that a number of control experiments are required when using liposome to mediated RNA transfection, and the possible consequences are discussed.

Detection of mRNA in Mammalian Cells with a Split Ribozyme Reporter

A Tetrahymena group I intron ribozyme reporter was split into two halves, and, in the presence of the target mRNA, the split reporter assembled into an active complex that trans-spliced and produced a reporter enzyme. We show that this split reporter can sense tumor-suppressor p53 mutant mRNA in mammalian cells. This system may open up new avenues for detecting and imaging RNA molecules in vivo.

Cover Picture: Detection of mRNA in Mammalian Cells with a Split Ribozyme Reporter (ChemBioChem 6/2006)

The cover picture shows a split RNA reporter strategy for detecting target mRNA in mammalian cells. A tetrahymena group I intron ribozyme reporter is split into two halves, each of which carries a part of the coding sequence of the reporter mRNA, a fragment of the ribozyme, and an antisense sequence complementary to a target mRNA. Both halves bind with the target mRNA and are assembled into an active complex that trans‐splices and produces a complete reporter mRNA. The reporter mRNA is subsequently translated into the reporter enzyme, which can be detected by fluorogenic substrates. Further details can be found in the article by J. Rao et al. on p. 925 ff.

RNA Interference: A Potential Therapeutic Tool for Silencing Splice Isoforms Linked to Human Diseases

Alternative splicing of precursor messenger RNAs (pre-mRNAs) is one of the most important sources of protein diversity in vertebrates. An estimated 35%-70% of human genes generate transcripts that are alternatively spliced, and defects in this process are linked to numerous human genetic diseases and various forms of cancer. The discovery that 21-23 nucleotide RNA duplexes, known as small interfering RNAs (siRNAs), can knockdown the homologous mRNAs in mammalian cells has revolutionized many aspects of drug discovery including down-regulation of disease-associated splicing isoforms. In addition, RNA interference (RNAi)-mediated silencing of splicing regulators has the potential to define the complex network of alternative splicing regulation and to analyze gene function. In this review, I first provide a brief introduction to mRNA splicing and its relationship to human diseases. This is followed by a brief overview of RNAi. Finally I discuss the therapeutic potential of RNAi in targeting disease-linked splicing isoforms.

A new function for nonsense-mediated mRNA-decay factors

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?