RNA Binding Proteins: New Concepts in Gene Regulation (Endocrine Updates)

Regulation of gene expression is thus critical to an organism's development. A gene is a stretch of DNA that encodes information. Genomic DNA consists of two antiparallel and reverse complementary strands, each having 5' and 3' ends.

The landscape of RNA-binding proteins

With respect to a gene, the two strands may be labeled the " template strand ," which serves as a blueprint for the production of an RNA transcript, and the " coding strand ," which includes the DNA version of the transcript sequence. Perhaps surprisingly, the "coding strand" is not physically involved in the coding process because it is the "template strand" that is read during transcription.

In eukaryotes, transcription is performed by three types of RNA polymerases, each of which needs a special DNA sequence called the promoter and a set of DNA-binding proteins— transcription factors —to initiate the process. When these residues are phosphorylated, the CTD binds to various protein factors that promote transcript maturation and modification. Transcription ends when the polymerase encounters a sequence called the terminator. While transcription of prokaryotic protein-coding genes creates messenger RNA mRNA that is ready for translation into protein, transcription of eukaryotic genes leaves a primary transcript of RNA pre-mRNA , which first has to undergo a series of modifications to become a mature mRNA.

These include 5' capping , which is set of enzymatic reactions that add 7-methylguanosine m 7 G to the 5' end of pre-mRNA and thus protect the RNA from degradation by exonucleases. Another modification is 3' cleavage and polyadenylation. The majority of eukaryotic pre-mRNAs consist of alternating segments called exons and introns.

During the process of splicing, an RNA-protein catalytical complex known as spliceosome catalyzes two transesterification reactions, which remove an intron and release it in form of lariat structure, and then splice neighbouring exons together. In certain cases, some introns or exons can be either removed or retained in mature mRNA.

This so-called alternative splicing creates series of different transcripts originating from a single gene. Because these transcripts can be potentially translated into different proteins, splicing extends the complexity of eukaryotic gene expression. Extensive RNA processing may be an evolutionary advantage made possible by the nucleus of eukaryotes.

In prokaryotes, transcription and translation happen together, whilst in eukaryotes, the nuclear membrane separates the two processes, giving time for RNA processing to occur. In most organisms non-coding genes ncRNA are transcribed as precursors that undergo further processing. While snoRNA part basepair with the target RNA and thus position the modification at a precise site, the protein part performs the catalytical reaction.

After being exported, it is then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer , which also initiates the formation of the RNA-induced silencing complex RISC , composed of the Argonaute protein. This is done either in the nucleoplasm or in the specialized compartments called Cajal bodies. In eukaryotes most mature RNA must be exported to the cytoplasm from the nucleus. Notably this includes all RNA types involved in protein synthesis. Every mRNA consists of three parts: The coding region carries information for protein synthesis encoded by the genetic code to form triplets.

Each triplet of nucleotides of the coding region is called a codon and corresponds to a binding site complementary to an anticodon triplet in transfer RNA. Transfer RNAs with the same anticodon sequence always carry an identical type of amino acid. Amino acids are then chained together by the ribosome according to the order of triplets in the coding region. In prokaryotes translation generally occurs at the point of transcription co-transcriptionally , often using a messenger RNA that is still in the process of being created.

In eukaryotes translation can occur in a variety of regions of the cell depending on where the protein being written is supposed to be. Major locations are the cytoplasm for soluble cytoplasmic proteins and the membrane of the endoplasmic reticulum for proteins that are for export from the cell or insertion into a cell membrane.

Proteins that are supposed to be expressed at the endoplasmic reticulum are recognised part-way through the translation process. This is governed by the signal recognition particle —a protein that binds to the ribosome and directs it to the endoplasmic reticulum when it finds a signal peptide on the growing nascent amino acid chain. The polypeptide folds into its characteristic and functional three-dimensional structure from a random coil. This polypeptide lacks any developed three-dimensional structure the left hand side of the neighboring figure.

Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins

Amino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein the right hand side of the figure known as the native state. The resulting three-dimensional structure is determined by the amino acid sequence Anfinsen's dogma. The correct three-dimensional structure is essential to function, although some parts of functional proteins may remain unfolded. Several neurodegenerative and other diseases are believed to result from the accumulation of misfolded proteins.

Enzymes called chaperones assist the newly formed protein to attain fold into the 3-dimensional structure it needs to function. Secretory proteins of eukaryotes or prokaryotes must be translocated to enter the secretory pathway. Newly synthesized proteins are directed to the eukaryotic Sec61 or prokaryotic SecYEG translocation channel by signal peptides. The efficiency of protein secretion in eukaryotes is very dependent on the signal peptide which has been used. Many proteins are destined for other parts of the cell than the cytosol and a wide range of signalling sequences or signal peptides are used to direct proteins to where they are supposed to be.

In prokaryotes this is normally a simple process due to limited compartmentalisation of the cell. However, in eukaryotes there is a great variety of different targeting processes to ensure the protein arrives at the correct organelle. Not all proteins remain within the cell and many are exported, for example, digestive enzymes , hormones and extracellular matrix proteins.

In eukaryotes the export pathway is well developed and the main mechanism for the export of these proteins is translocation to the endoplasmic reticulum, followed by transport via the Golgi apparatus. Regulation of gene expression refers to the control of the amount and timing of appearance of the functional product of a gene. Control of expression is vital to allow a cell to produce the gene products it needs when it needs them; in turn, this gives cells the flexibility to adapt to a variable environment, external signals, damage to the cell, and other stimuli.

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More generally, gene regulation gives the cell control over all structure and function, and is the basis for cellular differentiation , morphogenesis and the versatility and adaptability of any organism. Numerous terms are used to describe types of genes depending on how they are regulated; these include:. Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein.

The stability of the final gene product, whether it is RNA or protein, also contributes to the expression level of the gene—an unstable product results in a low expression level. In general gene expression is regulated through changes [24] in the number and type of interactions between molecules [25] that collectively influence transcription of DNA [26] and translation of RNA. Regulation of transcription can be broken down into three main routes of influence; genetic direct interaction of a control factor with the gene , modulation interaction of a control factor with the transcription machinery and epigenetic non-sequence changes in DNA structure that influence transcription.

Direct interaction with DNA is the simplest and the most direct method by which a protein changes transcription levels. Genes often have several protein binding sites around the coding region with the specific function of regulating transcription. There are many classes of regulatory DNA binding sites known as enhancers , insulators and silencers. The mechanisms for regulating transcription are very varied, from blocking key binding sites on the DNA for RNA polymerase to acting as an activator and promoting transcription by assisting RNA polymerase binding. The activity of transcription factors is further modulated by intracellular signals causing protein post-translational modification including phosphorylated , acetylated , or glycosylated.

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These changes influence a transcription factor's ability to bind, directly or indirectly, to promoter DNA, to recruit RNA polymerase, or to favor elongation of a newly synthesized RNA molecule. The nuclear membrane in eukaryotes allows further regulation of transcription factors by the duration of their presence in the nucleus, which is regulated by reversible changes in their structure and by binding of other proteins. More recently it has become apparent that there is a significant influence of non-DNA-sequence specific effects on transcription.

In general epigenetic effects alter the accessibility of DNA to proteins and so modulate transcription. DNA methylation is a widespread mechanism for epigenetic influence on gene expression and is seen in bacteria and eukaryotes and has roles in heritable transcription silencing and transcription regulation.

In eukaryotes the structure of chromatin , controlled by the histone code , regulates access to DNA with significant impacts on the expression of genes in euchromatin and heterochromatin areas. The majority of gene promoters contain a CpG island with numerous CpG sites.

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For example, in colorectal cancers about to genes are transcriptionally silenced by CpG island methylation see regulation of transcription in cancer. Transcriptional repression in cancer can also occur by other epigenetic mechanisms, such as altered expression of microRNAs.

In eukaryotes, where export of RNA is required before translation is possible, nuclear export is thought to provide additional control over gene expression. All transport in and out of the nucleus is via the nuclear pore and transport is controlled by a wide range of importin and exportin proteins.

Expression of a gene coding for a protein is only possible if the messenger RNA carrying the code survives long enough to be translated. In a typical cell, an RNA molecule is only stable if specifically protected from degradation. RNA degradation has particular importance in regulation of expression in eukaryotic cells where mRNA has to travel significant distances before being translated. In eukaryotes, RNA is stabilised by certain post-transcriptional modifications, particularly the 5' cap and poly-adenylated tail. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript.

These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs e. As of , the miRBase web site, [36] an archive of miRNA sequences and annotations, listed 28, entries in biologic species. The effects of miRNA dysregulation of gene expression seem to be important in cancer.

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders. Direct regulation of translation is less prevalent than control of transcription or mRNA stability but is occasionally used. Inhibition of protein translation is a major target for toxins and antibiotics , so they can kill a cell by overriding its normal gene expression control.

Protein synthesis inhibitors include the antibiotic neomycin and the toxin ricin. Once protein synthesis is complete, the level of expression of that protein can be reduced by protein degradation. There are major protein degradation pathways in all prokaryotes and eukaryotes, of which the proteasome is a common component. An unneeded or damaged protein is often labeled for degradation by addition of ubiquitin. Measuring gene expression is an important part of many life sciences , as the ability to quantify the level at which a particular gene is expressed within a cell, tissue or organism can provide a lot of valuable information.

For example, measuring gene expression can:. Similarly, the analysis of the location of protein expression is a powerful tool, and this can be done on an organismal or cellular scale. Investigation of localization is particularly important for the study of development in multicellular organisms and as an indicator of protein function in single cells. Ideally, measurement of expression is done by detecting the final gene product for many genes, this is the protein ; however, it is often easier to detect one of the precursors, typically mRNA and to infer gene-expression levels from these measurements.

May protect cells from apoptosis. Co-receptor of Wnt proteins. RYK is essential for Wnt-5a-dependent invasiveness in human glioma. Among interesting HuR targets, already known in other tissues, our data include i. To evaluate if this behavior occurs also in Nthy-ori Graphical representation of Nthy-ori The count distribution was normalized for the number of each region present. HuR is a RNA-binding protein that plays a major role in regulation of gene expression [ 10 ] and that can contribute to tumorigenesis [ 7 ].

The HuR function is controlled at multiple levels. HuR is able to positively regulate its own mRNA modulating cytoplasmic export, stabilization, and translation. Furthermore, HuR protein is subjected to several post-translational modification, i. Several data indicate that HuR expression and localization are modified in cancer cells. In particular, a very frequent observation is the increase of HuR cytoplasmic localization [ 4 , 16 ].

The relevance of HuR in tumorigenesis is highlighted by the evidence that this protein is currently explored as a target for anticancer treatment [ 17 , 18 ]. Nothing is known, so far, about HuR expression and function in thyroid follicular cells. Therefore, investigation on this protein could provide innovative information on molecular mechanisms contributing to thyroid cancer. Coherently with data obtained in different neoplasia [ 19 - 22 ], we show that HuR is over-expressed in thyroid cancer. When thyroid cancer cell lines were investigated, HuR over-expression was confirmed Figure 2.

Consistently with data obtained in tissues, both in Nthy-ori In this light, it is not surprising that HuR silencing reduced cell growth parameters in both cell lines. These results are coherent with available data on other cellular models; in fact, HuR silencing reduces cell growth of a wide range of cultured cells [ 25 - 28 ]. Colony formation assay of Nthy-ori Histogram representing the number of colonies per cell line. Cell viability of Nthy-ori All samples were run in quadruplicate. Considering HuR overexpression in thyroid cancer tissues as well as the results obtained by HuR silencing, indicating a HuR-dependent mechanism of thyroid cancer cell survival, this RBP might be consider an innovative therapeutic target for thyroid cancer.

How HuR silencing induces similar biological effects in Nthy-ori Two, not mutually exclusive, possibilities can be put forward. The first is that effects of HuR silencing are due to genes modified in both cell lines. The second possibility is that HuR silencing-derived biological effects are due to expression modification of distinct genes between the two cell lines. A major conclusion of our research is that in two cell lines originating from the same cell type thyroid follicular cells and both having a high proliferation rate, HuR shows significantly different spectra of both functional and interaction targets.

Here, functional targets are defined as those whose expression is modified by HuR silencing, while interaction targets are those delineated by the RIP-seq assay. Our experiments indicate that among functional targets, only 96 out of are shared by Nthy-ori It has been shown, in fact, that, when Jurkat cells are stimulated by the phorbol ester PMA, only among probes remain constant after 4 and 12 hours treatment [ 32 ].

Altogether, it could be envisaged that variation of HuR bound RNA species among cells with distinct biological properties is a common phenomenon. Our data represent a proof of concept that this variation exists between cells of the same origin but with different tumorigenic potential. Indeed, by comparison of RIP-seq data obtained in all cell lines, we observed a set of HuR-bound RNAs common for tumorigenic cell lines and not for non-tumorigenic cells.

An open question is to understand which phenotypic characteristics are responsible for this HuR behavior in non-tumorigenic and tumorigenic cells. Our immunohistochemical data indicate that the differences observed between the two cell lines are not due to a different protein localization Figure 2 , Panel C. Thus, a possible explanation of HuR plasticity, in terms of both functional and interaction targets, could be related to a difference its post-translational modifications in the two cell lines. HuR, indeed, is subjected to several post-translational modifications that influence its functions and its subcellular localization.

Nowadays more than kinds of RNA modifications have been identified and this discovery highlights the hypotesis that RNA modifications may act as epigenetic markers [ 33 ]. Wang and colleagues have demonstrated that HuR regulates the stability of many mRNAs in embryonic stem cells in a m 6 A-dependent manner, proving in particular, that the m 6 A methylation loss enhances HuR mRNA binding [ 35 ].

Our data indicate that in both cell lines, only a small fraction of functional targets is also interaction targets. In fact, in Nthy-ori In fact, for the yeast Puf3p protein, it has been shown that the expression of only 82 out of interaction targets was modified when transcriptomics of parental and Puf3p-deleted strains were compared [ 37 ]. We have to consider that these technological approaches are still new and major comprehension of their potential could improve the interpretation of data obtained so far.

However, the accuracy of our RNA-seq analysis is demonstrated by the consistency for all tested genes, whose expression has been evaluated by qPCR. Functional and interaction HuR target. In the circle overlapping area genes that were both Functional and Interaction HuR targets. Therefore, we could hypothesize that HuR knockdown caused a switch to an alternative gene expression program, in order to overcome the RBP absence.

Such a possibility is positively supported by our transcriptomic data. For example, miRb is downregulated upon HuR silencing and it is known that its downregulation is associated to promote apoptosis in PTCs [ 42 ]. In conclusion, our findings indicate that RBP targets may be different between cells with the same origin but with different aggressiveness. Therefore, investigation on this phenomenon could provide relevant information to understand the molecular derangements occurring in cancer cells.

A series of 12 normal thyroid glands NTs , 25 follicular adenomas FAs , 23 follicular thyroid carcinomas FTCs , 36 papillary thyroid carcinomas PTCs and 8 anaplastic thyroid carcinomas ATCs were selected from the files of the Institution of Anatomic Pathology of the University of Udine and the most representative blocks of each lesion were retrieved from the archive. All samples were diagnosed by referral pathologists of institutions and then reviewed by a single experienced pathologist, thus including only patients with a confirmed diagnosis.

In this study, we used 8 different thyroid cell lines: All cell lines have been validated by short tandem repeat and tested for being mycoplasma-free.

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A tissue microarray was created with representative tumor-bearing areas of the selected thyroid tissues. As for cell cultures, Nthy-ori This PBS—saponin solution was used for all subsequent washing steps. The tissue microarray and the cell cultures slides were incubated with primary rabbit polyclonal antiserum to HuR Millipore diluted 1: For reaction visualization, diaminobenzidine tetrahydrochloride was used as chromogen.

Using light microscopy, the entire section was scanned at high-power magnification x and nuclear immunostaining was evaluated by using the H-score method. The H-score is a well-established semi-quantitative evaluation of immunohistochemical data [ 43 ]. It is calculated by multiplying the percentage of immunoreactive cells and the intensity of the staining, which is graded as low score 1 , moderate score 2 or strong score 3.

Thus, the range of possible scores was from 0 to Each cellular location cytoplasm and nucleus was separately scored. H-score was determined by two experienced pathologists: Total protein extraction was performed as previously described [ 44 ].

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For nuclear and cytoplasmic protein extraction, Nthy-ori The membranes were then incubated overnight with rabbit polyclonal anti-HuR antibody 1: The day after, membranes were incubated for 2 h with anti-rabbit immunoglobulin coupled to peroxidase 1: The concentration of 5 nM was set performing dose-response studies data not shown.

The day before transfection, cells were plated in antibiotics-free medium. Cells were harvested 72 h after transfection and gene-silencing efficiency was evaluated by protein levels analysis. Signals for forward and side scatter and fluorescence were collected for 10 4 cells using the forward light scatter parameter as the master signal.

Data are expressed as mean fluorescence intensity FI values. Total RNA from Nthy-ori Both RNA samples and final libraries were quantified by using the Qubit 2. Raw sequence files were subjected to quality control analysis using FastQC http: In order to avoid low quality data, adapters were removed by Cutadapt [ 46 ] and lower quality bases were trimmed by ERNE [ 47 ]. For the analysis of differentially expressed genes, the quality-checked reads were processed using the TopHat version 2. Cuffdiff from the Cufflinks 2.

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For further analysis, we selected effective data using three criteria: Oligonucleotide primers were purchased from Sigma-Aldrich and their sequences are available upon request. In order to perform RIP assay, Nthy-ory The day after, samples were added to antibody-bead complexes and incubated overnight. Methylthiazolyldiphenyl-tetrazolium bromide MTT was applied to test cell viability.

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Plates were then incubated for 24, 48 and 72 h. All experiments were run in quadruplicate and cell viability was expressed as a fold change respect t0. Briefly, cells per plate were suspended in 4 ml of complete medium containing 0. Data are representative of three independent experiments. Siomi H, Dreyfuss G. RNA-binding proteins as regulators of gene expression. Curr Opin Genet Dev.

RNA-binding proteins and post-transcriptional gene regulation. Wurth L, Gebauer F. RNA-binding proteins, multifaceted translational regulators in cancer. Kechavarzi B, Janga SC. Dissecting the expressin landscape of RNA-binding proteins in human cancers. Abdelmohsen K, Gorospe M. Posttranscriptional regulation of cancer traits by HuR. Int J Mol Sci. Govindaraju S, Lee BS. World J Biol Chem. Srikantan S, Gorospe M. HuR function in disease. Increasing incidence of differentiated thyroid cancer in the United States, Emerging strategies for managing differentiated thyroid cancers refractory to radioiodine.

Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression. Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast carcinoma.