Coding Sequences Of mRNAs Research Articles

Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells.

Simple SummarymiRNAs are omnipresent short non-coding RNA molecules, which post-transcriptionally fine-tune the expression of most protein-coding genes in health and disease. In cancer, miRNAs have been described to directly regulate the amounts of targeted tumor suppressors and oncogenes. Although their canonical mechanism of action is well understood, the correct prediction of miRNA target genes is still a challenge. We describe here the unbiased investigation of the miRNA targetome in cancer (melanoma) cells using a technique, which allows to physically link the miRNA to its target gene. The herein identified miRNA-target interactions reveal further layers of complexity of post-transcriptional gene regulation in cancer cells and shed new light on miRNA-target gene interactions.MicroRNAs are key post-transcriptional gene regulators often displaying aberrant expression patterns in cancer. As microRNAs are promising disease-associated biomarkers and modulators of responsiveness to anti-cancer therapies, a solid understanding of their targetome is crucial. Despite enormous research efforts, the success rates of available tools to reliably predict microRNAs (miRNA)-target interactions remains limited. To investigate the disease-associated miRNA targetome, we have applied modified cross-linking ligation and sequencing of hybrids (qCLASH) to BRAF-mutant melanoma cells. The resulting RNA-RNA hybrid molecules provide a comprehensive and unbiased snapshot of direct miRNA-target interactions. The regulatory effects on selected miRNA target genes in predicted vs. non-predicted binding regions was validated by miRNA mimic experiments. Most miRNA–target interactions deviate from the central dogma of miRNA targeting up to 60% interactions occur via non-canonical seed pairing with a strong contribution of the 3′ miRNA sequence, and over 50% display a clear bias towards the coding sequence of mRNAs. miRNAs targeting the coding sequence can directly reduce gene expression (miR-34a/CD68), while the majority of non-canonical miRNA interactions appear to have roles beyond target gene suppression (miR-100/AXL). Additionally, non-mRNA targets of miRNAs (lncRNAs) whose interactions mainly occur via non-canonical binding were identified in melanoma. This first application of CLASH sequencing to cancer cells identified over 8 K distinct miRNA–target interactions in melanoma cells. Our data highlight the importance non-canonical interactions, revealing further layers of complexity of post-transcriptional gene regulation in melanoma, thus expanding the pool of miRNA–target interactions, which have so far been omitted in the cancer field.

SMN-primed ribosomes modulate the translation of transcripts related to spinal muscular atrophy

The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains enigmatic. We demonstrate that Survival Motor Neuron (SMN) protein, loss of which causes the neuromuscular disease spinal muscular atrophy (SMA), binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs which are enriched for translational enhancer sequences in the 5’UTR and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs which encode proteins relevant to SMA pathogenesis.

Eukaryotic Translation Elongation is Modulated by Single Natural Nucleotide Derivatives in the Coding Sequences of mRNAs.

RNA modifications are crucial factors for efficient protein synthesis. All classes of RNAs that are involved in translation are modified to different extents. Recently, mRNA modifications and their impact on gene regulation became a focus of interest because they can exert a variety of effects on the fate of mRNAs. mRNA modifications within coding sequences can either directly or indirectly interfere with protein synthesis. In order to investigate the roles of various natural occurring modified nucleotides, we site-specifically introduced them into the coding sequence of reporter mRNAs and subsequently translated them in HEK293T cells. The analysis of the respective protein products revealed a strong position-dependent impact of RNA modifications on translation efficiency and accuracy. Whereas a single 5-methylcytosine (m5C) or pseudouridine (Ψ) did not reduce product yields, N1-methyladenosine (m1A) generally impeded the translation of the respective modified mRNA. An inhibitory effect of 2′O-methlyated nucleotides (Nm) and N6-methyladenosine (m6A) was strongly dependent on their position within the codon. Finally, we could not attribute any miscoding potential to the set of mRNA modifications tested in HEK293T cells.

Small RNA sequencing revealed dysregulated piRNAs in Alzheimer's disease and their probable role in pathogenesis.

PIWI-interacting RNAs (piRNAs), ∼23-36 nucleotide-long small non-coding RNAs, earlier believed to be germline-specific, have now been identified in somatic cells including neural cells. However, piRNAs have not yet been studied in the human brain (HB) and Alzheimer's disease (AD)-affected brain. In this study, by next-generation small RNA sequencing, 564 and 451 piRNAs were identified in the HB and AD-affected brain respectively. The majority of the neuronal piRNAs have intronic origin wherein primary piRNAs are mostly from the negative strand. piRNAs originating from the coding sequence of mRNAs and tRNAs are highly conserved compared to other genomic contexts. We found 1923 mRNAs significantly down-regulated in AD as the predicted targets of 125 up-regulated piRNAs. The filtering of targets based on our criteria coupled with pathway enrichment analysis of all the predicted targets resulted in five most significant AD-associated pathways enriched with four genes (CYCS, LIN7C, KPNA6, and RAB11A) found to be regulated by four piRNAs. The qRT-PCR study verified the reciprocal expression of piRNAs and their targets. This study provides the first evidence of piRNAs in the HB and AD which will provide the foundation for future studies to unravel the regulatory role of piRNAs in the human brain and associated diseases. The sequencing data have been submitted to the GEO database (Accession no. GSE85075).

Experimental Genome-Wide Determination of RNA Polyadenylation in Chlamydomonas reinhardtii.

The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types—Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth.

Deep sequencing identifies noncanonical editing of Ebola and Marburg virus RNAs in infected cells.

ABSTRACTDeep sequencing of RNAs produced by Zaire ebolavirus (EBOV) or the Angola strain of Marburgvirus (MARV-Ang) identified novel viral and cellular mechanisms that diversify the coding and noncoding sequences of viral mRNAs and genomic RNAs. We identified previously undescribed sites within the EBOV and MARV-Ang mRNAs where apparent cotranscriptional editing has resulted in the addition of non-template-encoded residues within the EBOV glycoprotein (GP) mRNA, the MARV-Ang nucleoprotein (NP) mRNA, and the MARV-Ang polymerase (L) mRNA, such that novel viral translation products could be produced. Further, we found that the well-characterized EBOV GP mRNA editing site is modified at a high frequency during viral genome RNA replication. Additionally, editing hot spots representing sites of apparent adenosine deaminase activity were found in the MARV-Ang NP 3′-untranslated region. These studies identify novel filovirus-host interactions and reveal production of a greater diversity of filoviral gene products than was previously appreciated.

MicroRNA-200c modulates the expression of MUC4 and MUC16 by directly targeting their coding sequences in human pancreatic cancer.

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

RNase III-Mediated Silencing of a Glucose-Dependent Repressor in Yeast

Members of the RNase III family are found in all species examined with the exception of archaebacteria, where the functions of RNase III are carried out by the bulge-helix-bulge nuclease (BHB). In bacteria, RNase III contributes to the processing of many noncoding RNAs and directly cleaves several cellular and phage mRNAs. In eukaryotes, orthologs of RNase III participate in the biogenesis of many miRNAs and siRNAs, and this biogenesis initiates the degradation or translational repression of several mRNAs. However, the capacity of eukaryotic RNase IIIs to regulate gene expression by directly cleaving within the coding sequence of mRNAs remains speculative. Here we show that Rnt1p, a member of the RNase III family, selectively inhibits gene expression in baker's yeast by directly cleaving a stem-loop structure within the mRNA coding sequence. Analysis of mRNA expression upon the deletion of Rnt1p revealed an upregulation of the glucose-dependent repressor Mig2p. Mig2p mRNA became more stable upon the deletion of Rnt1p and resisted glucose-dependent degradation. In vitro, Rnt1p cleaved Mig2p mRNA and a silent mutation that disrupts Rnt1p signals blocked Mig2p mRNA degradation. These observations reveal a new RNase III-dependent mechanism of eukaryotic mRNA degradation.

Molecular Cloning and Characterization of a Translational Inhibitory Protein That Binds to Coding Sequences of Human Acid β-Glucosidase and Other mRNAs

Acid β-glucosidase (GCase) is the enzyme deficient in Gaucher disease, a prototypical inherited metabolic error for enzyme and gene therapy. An 80-kDa cytoplasmic protein, termed TCP80, was found to inhibit GCase mRNA translation in mammalian cells by binding to RNA-coding regions. The TCP80 cDNA was cloned by screening an expression library with the GCase-coding region RNA. The cDNA sequence was nearly identical to those for M-phase phosphoprotein (MPP4; 99%) and for the IL-2 enhancer binding protein (NF90; 96%). Expression of the carboxy-terminal third, TCP30, showed it to be an RNA-binding protein that bound to a 184-nt fragment of GCase-coding sequence near the 5′ end of the mature mRNA. When added to reactions, a large molar excess of TCP30 diminished the translation inhibition of GCase RNA by cytoplasmic TCP80. TCP50, expressed from the NH2-terminal two-thirds of TCP80, did not bind to nor inhibit the translation of GCase RNA. Reconstitution of in vitro translation inhibition of GCase RNA required intact human TCP80 heterologously expressed in insect cells. Time course analyses show that TCP80 functions at the initiation phase of GCase mRNA translation, probably by inhibiting its binding to polysomes. Seven additional RNAs were isolated by specific binding to TCP30 including those for aldolase B, complement protein 8 γ-subunit, fibronectin receptor β1, ABL, lactate dehydrogenase A, fibrinogen γ-chain, and peroxisomal proliferator-activated receptor α. In vitro translation of their RNAs was inhibited by TCP80. These studies show that TCP80 has RNA-binding (TCP30) and inhibitory (TCP50) domains that function to modulate translation of several mRNAs. TCP80 is likely identical to MPP4 and NF90, but has previously undescribed roles in cellular function.