Interactive Fly, Drosophila

elav


EVOLUTIONARY HOMOLOGS (part 2/2)

ELAV homologs target 3' UTRs of mRNAs

Mammalian cDNAs encoding a rat (Rel-N1) and a human (Hel-N1) neuronal RNA-binding protein have been cloned and characterized with respect to tissue specificity, neuroanatomical localization, and RNA binding specificity. Both proteins are highly similar to the product of the Drosophila elav gene. However, in situ hybridization of rat tissues demonstrate more restricted expression of Rel-N1 mRNA within a subset of neurons of the hippocampus, cortex, and other regions of the gray matter, but not in glial cells or white matter. In vitro RNA binding experiments demonstrate that Hel-N1 can bind to the 3' untranslated region (3' UTR) of Id mRNA, a transcript that encodes a helix-loop-helix transcriptional repressor that is abundantly expressed in undifferentiated neural precursors. Sequences characterized for Hel-N1 binding are also abundantly present in the 3' UTR of the Drosophila Extramacrochaetae mRNA, which encodes an Id homolog. Thus, a potential link was identified between a neuronal 3' UTR RNA-binding protein and regulatory transcription factors involved in neural development (King, 1994).

Both Hel-N1 and Hel-N2 bind to A+U-rich 3' untranslated regions of a variety of growth-related mRNAs in vitro. In medulloblastoma cells derived from childhood brain tumors, Hel-N1 and Hel-N2 are mainly expressed in the cytoplasm, but are detectable in the nucleus. Both proteins are associated with polysomes and can be UV-crosslinked to poly(A)+ mRNA in cell extracts. In the cytoplasm the Hel-N1 protein family resides in granular structures that may contain multiple protein molecules bound to each mRNA. Evidence supporting this multimeric ribonucleoprotein (RNP) model includes in vitro reconstitution and competition experiments in which addition of a single RRM (RRM3) can alter complex formation. As in medulloblastoma cells, the Hel-N1 protein family is present in granular particles in the soma and the proximal regions of dendrites of cultured neurons, and colocalizes with ribosomes. In addition, expression of the Hel-N1 protein family is up-regulated during neuronal differentiation of embryonic carcinoma P19 cells. These data suggest that the Hel-N1 protein family is associated with the translational apparatus and implicated in both mRNA metabolism and neuronal differentiation. Furthermore, it is possible that these proteins participate in mRNA homeostasis in the dendrites and soma of mature neurons (Gao, 1996).

3T3-L1 preadipocytes ectopically expressing the mammalian RNA-binding protein Hel-N1 express up to 10-fold more glucose transporter (GLUT1) protein and exhibit elevated rates of basal glucose uptake. ELAV proteins are known to bind in vitro to short stretches of uridylates in the 3' untranslated regions (3'UTRs) of unstable mRNAs encoding growth-regulatory proteins involved in transcription and signal transduction. GLUT1 mRNA also contains a large 3'UTR with a U-rich region that binds specifically to Hel-N1 in vitro. Analysis of the altered GLUT1 expression at the translational and posttranscriptional levels suggests a mechanism involving both mRNA stabilization and accelerated formation of translation initiation complexes. These findings are consistent with the hypothesis that the Hel-N1 family of proteins modulate gene expression at the level of mRNA in the cytoplasm (Jain, 1997).

The GLUT1 glucose transporter has an extensive 3' UTR that is AU-rich reminiscent of the 3'UTRs of an oncogene mRNA. An in vitro RNA binding assay using Hel-N1 demonstrates binding to a specific portion of the GLUT1 3'UTR. Analysis of the folding pattern of this region depictes the retention of a stem loop structure, wherein the loop is composed of a stretch of uridylates. To further analyze the potential function of Hel-N1, stable transfectants were made in the 3T3-L1 cell line. The transfectants have been characterized, and the presence of the Hel-N1 DNA and protein verified. Data indicate Hel-N1 is a ligand for GLUT1; its binding affects the stability and translatability of the GLUT1 message (Jain, 1995).

The RNA binding specificity was examined of Hel-N1, a human neuron-specific RNA-binding protein that contains three RNA recognition motifs. Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrate that Hel-N1 binds and forms multimers with c-myc 3' UTR mRNA and requires, at a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrates that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. Hel-N1 is reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA (Levine, 1993).

Turnover of labile mRNAs is thought to be mediated in part by the interactions of trans-acting factors with elements withing the 3' untranslated region. Neuronal and non-neuronal cells established from neuroblastoma tumors differ in N-myc mRNA levels (See Drosophila MYC). There are two distinct regions within the 3'-UTR of N-myc mRNA that bind a 40kDA protein complex present in non-neuronal cells but absent from neuronal cells. The N-myc binding protein is identified as a member of the ELAV-like family of RNA-binding proteins. It is likely that the ELAV-like mRNA-binding protein acts to stabilize the mRNA, and potentially regulates N-myc mRNA turnover (Chagnovich, 1996).

Mouse-Musashi-1 RNA-recognition motif is 60% identical to Xenopus NRP-1 and 95% identical to Drosophila Musashi. In the C-terminal half of m-Msi-1, which does not contain an RNA-recognition motif, m-Msi-1 shows a 30% homology to Drosophila MSI. m-Msi-1 is preferentially expressed in neural tissues, especially mitotically active neural precursor cells within the CNS. The m-Msi-1 expressing cells overlap the major population of nestin intermediate filament positive cells. Lineage analysis using single neuroepithelial cell culture systems reveal that m-Msi-1 is highly enriched in CNS cells, precursors of neuronal and glial cells. However, m-Msi-1 expression is rapidly down-regulated during neural differentiation. Expression of m-Msi-1 protein shows a pattern complementary to that of another mammalian RNA-binding protein, Hu (a mammalian homolog of the Drosophila neuron-specific RNA binding protein ELAV). Hu is exclusively expressed in postmitotic neurons in the CNS. In vitro studies indicate that these two proteins have distinct RNA target species. Therefore, it is likely that m-Msi-1 and Hu have distinct roles in neurogenesis that are relevant to those of Drosophila MSI and ELAV, respectively (Sakakibara, 1996).

The Elav-like proteins are specific mRNA-binding proteins that regulate mRNA stability. The neuronal members of this family (HuD, HuC, and Hel-N1) are required for neuronal differentiation. In this report, using purified HuD protein, a high affinity HuD binding site has been localized to a 42-nucleotide region within a U-rich tract in the 3'-untranslated region p21(waf1) mRNA. The binding of HuD to this site is readily displaced by an RNA oligonucleotide encoding the HuD binding site of c-fos. The sequence of this binding site is well conserved in human, mouse, and rat p21(waf1) mRNA. p21(waf1) is an inhibitor of cyclin-dependent kinases and proliferating cell nuclear antigen and induces cell cycle arrest at G1/S, a requisite early step in cell differentiation. The identification of an Elav-like protein binding site in the 3'-untranslated region of p21(waf1) provides a novel link between the induction of differentiation, mRNA stability, and the termination of the cell cycle (Joseph, 1998).

The Elav-like proteins are specific mRNA binding proteins that are required for cellular differentiation. They contain three characteristic RNP2/RNP1-type RNA binding motifs. The first and second RNA binding domains bind to AU-rich elements in the 3'-UTR of mRNA. Elav-like proteins are shown to exhibit poly(A) binding activity. This activity is distinct from poly(A) binding activities. The Elav-like proteins specifically bind to long chain poly(A) tails. The third RNA binding domain encompasses this poly(A) binding activity. Elav-like proteins can bind simultaneously to the AU-rich element and to the poly(A) tail (Ma, 1997).

An in vitro mRNA stability system using HeLa cell cytoplasmic S100 extracts and exogenous polyadenylated RNA substrates was developed that reproduces regulated aspects of mRNA decay. The addition of cold poly(A) competitor RNA activates both a sequence-specific deadenylase activity in the extracts as well as a potent, ATP-dependent ribonucleolytic activity. The rates of both deadenylation and degradation are up-regulated by the presence of a variety of AU-rich elements in the body of substrate RNAs. Competition analyses demonstrates that trans-acting factors are required for RNA destabilization by AU-rich elements. The ~30-kD ELAV protein HuR specifically binds to RNAs containing an AU-rich element derived from the TNF-alpha mRNA in the in vitro system. However, interaction of HuR with AU-rich elements is not associated with RNA destabilization. Interestingly, recombinant ELAV proteins specifically stabilize deadenylated intermediates generated from the turnover of AU-rich element-containing substrate RNAs. These data suggest that mammalian ELAV proteins play a role in regulating mRNA stability by influencing the access of degradative enzymes to RNA substrates (Ford, 1999).

Human members of the Elav family, referred to as Elav-like proteins (ELPs), include HuC, HuD, Hel-N1 and HuR. These proteins bind to AU-rich elements in the 3'-untranslated regions (3'-UTRs) of many growth-related mRNAs, including c-myc and VEGF, and may participate in regulating the stability of these transcripts. An enzyme-linked immunosorbent assay (ELISA) has been developed that can rapidly assess the RNA-protein-binding properties of ELPs. With this assay, it is demonstrated that HuC and HuD bind to the VEGF 3'-UTR regulatory segment (VRS) and to the c- myc 3'-UTR in a specific and concentration-dependent pattern, with both proteins showing a greater affinity for the VRS. Further analysis of the VRS indicates that the binding affinity is greater for the 3'-end where the majority of AU motifs reside. Binding to the VRS can be competed by both proteins as well as a poly(U) ribohomopolymer. The binding can not be competed by other ribohomopolymers or serum from patients with high titer anti-HuD antibodies. In summary, this assay provides a rapid analysis of ELP-RNA binding that can be utilized for further characterization of RNA-binding properties and for identification of competitor molecules for in vivo functional analysis of ELPs (King, 2000a).

Human ELAV proteins are implicated in cell growth and differentiation via regulation of mRNA expression in the cytoplasm. In human embryonic teratocarcinoma (hNT2) cells transfected with the human neuronal ELAV-like protein, Hel-N1, neurites form, yet cells are not terminally differentiated. Cells in which neurite formation is associated with Hel-N1 overexpression also express increased levels of endogenous neurofilament M (NF-M) protein, which distributes along the neurites. However, steady-state levels of NF-M mRNA remain similar whether or not hNT2 cells are transfected with Hel-N1. These findings suggest that turnover of NF-M mRNA is not affected by Hel-N1 expression, despite the fact that Hel-N1 can bind to the 3' UTR of NF-M mRNA and is found directly associated with NF-M mRNA in transfected cells. Analysis of the association of NF-M mRNA with the translational apparatus in Hel-N1 transfectants shows nearly complete recruitment to heavy polysomes, indicating that Hel-N1 causes an increase in translational initiation. These results suggest that the stability and/or translation of AU-rich element-containing mRNAs can be independently regulated by the ELAV protein, Hel-N1, depending upon sequence elements in the 3' UTRs and upon the inherent turnover rates of the mRNAs that are bound to Hel-N1 in vivo (Antic, 1999).

h1ARE, a 57-nucleotide adenosine- and uridine-rich RNA instability element in the human papillomavirus type 1 late 3' untranslated region, interacts specifically with three nuclear proteins that fail to bind to an inactive mutant RNA. Two of these have been identified as the heterogeneous ribonucleoproteins C1 and C2, whereas the third, a 38-kDa, poly(U) binding protein (p38), has remained unidentified. Here it is shown that partially purified p38 reacts with a monoclonal antibody raised against the recently identified Elav-like HuR protein, indicating that p38 is the HuR protein. Indeed, recombinant glutathione S-transferase (GST)-HuR protein binds specifically to sites within the h1ARE. Determination of the apparent Kd value of GST-HuR for the h1ARE and the inactive mutant thereof reveal that GST-HuR binds with a more than 50-fold-higher affinity to the wild-type sequence. Therefore, the binding affinity of GST-HuR for the wild-type and mutant h1AREs correlate with their inhibitory activities in transfected cells, strongly suggesting that the HuR protein is involved in the posttranscriptional regulation of human papillomavirus type 1 late-gene expression (Sokolowski, 1999).

AU-rich elements (AREs) located in the 3' untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the Embryonic lethal abnormal vision (Elav) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. A mouse monoclonal antibody, 3A2, recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that stabilizes ARE-containing mRNAs. At 37 degrees C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)plus RNA are predominantly cytoplasmic rather than nuclear. This provides evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12%-15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A) plus RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. These differences are interpreted to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of the other two proteins (Gallouzi, 2000).

Cytokine stimulation of human DLD-1 cells results in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there is a basal transcription of the NOS II gene, which does not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR show a significantly reduced luciferase activity. The Elav-like protein HuR binds with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduces the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiates the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA (Rodriguez-Pascual, 2000).

3'-untranslated regions of various mRNAs have been shown to contain sequence motifs that control mRNA stability, translatability, and efficiency of translation as well as intracellular localization. Protein binding regions have been sought for the long and highly conserved 3'UTR of the mRNA coding for neurofibromin, a well-known tumor suppressor protein, whose genetic deficiency causes the autosomal dominant disease neurofibromatosis type 1 (NF1). Five RNA fragments are able to undergo specific binding to proteins from cell lysates (NF1-PBRs, NF1-protein-binding regions). Additionally the Elav-like protein HuR binding to NF1-PBR1, has been identified. HuR interacts with AU-rich elements in the 3'UTR of many protooncogenes, cytokines, and transcription factors, thereby regulating the expression of these mRNAs on the posttranscriptional level. Transfection assays with a CAT reporter construct reveal reduced expression of the reporter, suggesting that HuR may be involved in the fine-tuning of the expression of the NF1 gene (Haeussler, 2000).

p27Kip1 restrains cell proliferation by binding to and inhibiting cyclin-dependent kinases. To investigate the mechanisms of p27 translational regulation, a complete p27 cDNA was isolated and an internal ribosomal entry site (IRES) was identified, located in the p27 5'UTR. The IRES allows for efficient p27 translation under conditions where cap-dependent translation is reduced. Searching for possible regulators of IRES activity the neuronal ELAV protein HuD was identified as a specific binding factor of the p27 5'UTR. Increased expression of HuD or the ubiquitously expressed HuR protein specifically inhibits p27 translation and p27 IRES activity. Consistent with an inhibitory role of Hu proteins in p27 translation, siRNA mediated knockdown of HuR induces endogenous p27 protein levels as well as IRES-mediated reporter translation and leads to cell cycle arrest in G1 (Kullmann, 2002).

What is the physiologic role of the IRES? Analysis of the effect of PI3 kinase inhibition on p27 translation suggests that p27 translation can be mediated in large part via its IRES element. It is speculated that the IRES serves to sustain p27 synthesis under unfavorable growth conditions, when overall cap-dependent protein synthesis is reduced and persisting p27 expression is required, for example, in the case of some viral infections. In addition, maintenance of translation under stress conditions or during quiescence may relay on IRES elements. For example, VEGF is thought to be translated by internal ribosomal entry under hypoxia, when overall protein synthesis is compromised. Interestingly hypoxia has also been shown to induce p27 protein levels. Efficient translation of the Cdk inhibitor under these conditions may therefore depend on its IRES element (Kullmann, 2002).

The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3'-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation; this process depends on the size of the poly(A) tail. Using a polysome-based in vitro mRNA decay assay, it was found that addition of recombinant HuD protein to the system increases the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect is caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization is specific for GAP-43 mRNA containing the HuD binding element in the 3'-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, it was found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). It is concluded that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA (Beckel-Mitchener, 2002).

Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. Nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching approximately 3.5-fold by 72 h. Given that the AChE 3'-untranslated region (UTR) contains an AU-rich element, focus was placed on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, these studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3'-UTR shows that this 3'-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots reveal an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrate that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a key trans-acting factor in these events (Deschenes-Furry, 2003).

Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system; HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays reveal three HuD-binding sequences in the 3'-untranslated region of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3'-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. These results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3'-UTR, which prolongs the mRNA lifetime and increases protein level (Cuadrado, 2002).

MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. An ELAV-like protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. The ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells has been characterized and the functional consequences of its interactions with the ELAV protein HuD has been examined. At least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene (Manohar, 2002).

The Wnt/ß-Catenin --> Pitx2 pathway controls the turnover of Pitx2 and other unstable mRNAs: Pitx2 binds to and regulates HuR

The Wnt/β-catenin pathway rapidly induces the transcription of the cell-type-restricted transcription factor Pitx2 that is required for effective cell-specific proliferation activating growth-regulating genes. Pitx2 mRNA displays a rapid turnover rate and activation of the Wnt/β-catenin pathway stabilizes Pitx2 mRNA as well as other unstable mRNAs, including c-Jun, Cyclin D1, and Cyclin D2, encoded by critical transcriptional target genes of the same pathway. The data indicate that Pitx2 mRNA stabilization is due to a reduced interaction of Pitx2 3'UTR with the destabilizing AU-rich element (ARE) binding proteins (BPs) KSRP and TTP as well as to an increased interaction with a stabilizing ARE-BP, HuR [ELAV-like 1 (Hu antigen R)]. Pitx2 itself is a mediator of Wnt/β-catenin-induced mRNA stabilization. These previous and present data support the hypothesis that a single pathway can coordinately regulate sequential transcriptional and posttranscriptional events leading to an integrated functional gene regulatory network (Briata, 2003).

Many tissue-restricted transcription factors mediate crucial steps during development, functioning as the distal targets of different classes of regulatory signaling pathways. One of these, the Wnt signaling cascade, controls organogenesis by inducing a wide range of responses from cell proliferation to cell fate determination and terminal differentiation. Extracellular Wnt signals activate the cytoplasmic protein Dishevelled (Dvl) that, in turn, inhibits the constitutive proteasomal destruction of β-catenin. As a result, β-catenin accumulates in the nucleus, associates with TCF/LEF transcription factors, and TCF/LEF target genes become transiently activated (Briata, 2003).

Pitx2 gene, which encodes a transcription factor belonging to the bicoid family and exerts a crucial role during mammalian development, is a LEF1 target gene. A Wnt/Dvl/β-catenin->Pitx2 pathway has been described that mediates cell-type-specific proliferation during cardiac outflow tract and pituitary gland development. Once induced by Wnt signaling, Pitx2 is required for cell-type-specific proliferation and directly activates specific growth-regulating genes, such as Cyclin D1, Cyclin D2, and c-Myc. The rapid induction of Pitx2 by Wnt as well as the observation that the expression of Pitx2 is tightly regulated in time and space during development, led to the hypothesis that additional levels at which Pitx2 expression can be modulated might exist (Briata, 2003 and references therein).

It is a rising concept that most genes are regulated by multiple mechanisms, the sum of which dictates the unique expression pattern of a gene under certain conditions. Several examples suggest that gene transcription and mRNA degradation rates are coordinately regulated to allow temporal modulation of gene expression. The rate of mRNA turnover not only determines the rate of disappearance of mRNA but also its induction. mRNAs with short half-lives respond to changes in transcription more rapidly than those that are relatively stable, thus contributing to rapid changes in the pattern of cellular gene expression in response to changing environmental or developmental cues. Inherently unstable mRNAs include those encoding oncoproteins, cytokines, and cell cycle-regulated proteins. Furthermore, during early Drosophila development, several genes undergo dramatic changes in abundance and in spatial distribution. To achieve these rapid changes, multiple regulatory mechanisms exist that include both transcriptional control and regulation of mRNA processing (Briata, 2003 and references therein).

Rapid degradation of mRNAs requires at least three components, (1) an instability element, such as the adenylate/uridylate-rich element (ARE) located in the 3'-untranslated region (UTR); (2) certain ARE binding proteins (ARE-BPs), and (3) an enzyme, the exosome. RNA decay is compromised by the removal of any of these components. AREs have been recognized as potent destabilizing elements in a wide variety of short-lived mRNAs. AREs are grouped into three classes according to their sequence features and RNA decay characteristics. Class I AREs contain 1 to 3 scattered copies of the pentanucleotide AUUUA embedded within a U-rich region, and are found in the c-Fos and c-Myc mRNAs. Class II AREs contain multiple overlapping copies of the AUUUA motif, and are found in cytokine mRNAs. Class III AREs, such as the one in c-Jun mRNA, lack the hallmark AUUUA pentanucleotide but present U-rich sequences. Some ARE-BPs have been proven to possess destabilizing activity on ARE-RNAs (TTP, BRF1, KSRP), while another (HuR) has been demonstrated to stabilize target transcripts. AUF1 has a dual role in ARE-mediated mRNA decay, functioning either as a destabilizing or a stabilizing factor depending on the cell type. The described ARE-BPs are required for regulation of class I and II ARE-RNAs. However, it is unclear whether the same ARE-BPs are involved in the control of class III ARE-RNAs. The exosome is a multisubunit particle, containing nine 3'-to-5' exoribonucleases and some ARE-BPs, that rapidly degrade ARE-RNAs (Briata, 2003 and references therein).

This study reports an unexpected mechanism controlling Pitx2 gene expression. Pitx2 mRNA is rapidly degraded due to the presence of destabilizing elements in both its coding sequence and 3'UTR. Activation of the Wnt/β-catenin pathway in pituitary cells induces a strong stabilization of Pitx2 mRNA as well as of Cyclins D1 and D2, and c-Jun mRNAs, which are known to be transcriptional targets of the Wnt pathway. Pitx2 mRNA stabilization correlates with a change in the pattern of interaction of both destabilizing and stabilizing ARE-BPs with Pitx2 3'UTR, with Pitx2 itself modulating the turnover of unstable mRNAs subsequent to Wnt/β-catenin activation (Briata, 2003).

A model is proposed that integrates a view of how Wnt signaling can regulate the expression of target genes in pituitary-derived cells. Wnt activation rapidly induces, through LEF1, the transcription of Pitx2 and of additional target genes, including c-Jun, Cyclin D1, and Cyclin D2, through either TCF/LEF or Pitx2. Wnt activation regulates the expression of the same target genes affecting their mRNA turnover rates. Once induced, Pitx2 itself plays a central role in the stabilization of its own transcript and in the turnover control of other unstable transcripts (Briata, 2003).

The results suggest a direct role of Pitx2 in controlling HuR function: (1) LiCl treatment of αT3-1 cells strongly increases cytoplasmic levels of Pitx2; (2) Pitx2 interacts with HuR; (3) a Pitx2 mutant unable to exert cell-type-specific proliferation control does not interact with HuR and does not reconstitute RNA stabilization in αPitx2-immunodepleted S100. Furthermore, this mutant blocks LiCl effect on mRNA stability functioning as a Pitx2 dominant negative in αT3-1 cells. (4) Neither recombinant nor endogenous Pitx2 is able to directly interact with ARE mRNA, ruling out the possibility of an RNA binding-dependent function of Pitx2 in mRNA stabilization. It is tempting to speculate that Pitx2 plays a role in modulating the cytoplasmic concentration of HuR and, consequently, its in vitro and in vivo binding activity to ARE-mRNAs. However, on the basis of the data, it is suggested that Pitx2 regulates additional events that control mRNA stability. Pitx2 immunodepletion from LiCl-treated S100 removes less than 50% of HuR present in the extracts while it causes a complete destabilization of Pitx2 3'UTR RNA. Conversely, complete HuR immunodepletion from LiCl-treated S100 (that removes less than 50% of Pitx2) does not completely destabilize Pitx2 3'UTR RNA. Altogether these results suggest that HuR is not the only target of Pitx2-mediated mRNA stabilization (Briata, 2003).

Regulating a rate-limiting step is an efficient way to control the overall rate of a multistep process. However, there is a limit to the level of regulation that can be achieved by controlling a single step; there is no point in increasing the rate of one step if another soon becomes rate limiting. Therefore, eukaryotes have developed methods to regulate the expression of their proteins at multiple levels in a coordinated fashion. The mechanisms used largely depend on the level of regulation required for proper gene function and are selected through evolution. Genes whose expression must be rapidly and tightly controlled tend to be quickly transcribed and translated, and their mRNAs and proteins have short half-lives. These data add a further level to the definition of a multistep regulation by the Wnt/β-catenin pathway that enhances the expression of selected genes by, at least, two independent and coordinated mechanisms. In a sense, these events convert Pitx2 from a rapidly induced gene to a more stable regulator of cell-type-specific gene function (Briata, 2003).

ELAV homologs and nucleocytoplasmic shuttling

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3' untranslated regions and has been shown to shuttle between the nucleus and cytoplasm. A shuttling sequence has been identified in HuR that also possesses transcription-dependent nuclear localization signal activity. It is proposed that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment (Fan, 1998b).

AU-rich elements (AREs), present in the 3' untranslated regions of many protooncogene, cytokine, and lymphokine messages, target these mRNAs for rapid degradation. HuR, a ubiquitously expressed member of the Elav (Embryonic lethal abnormal vision) family of RNA binding proteins, selectively binds AREs and stabilizes ARE-containing mRNAs in transiently transfected cells. Four mammalian proteins have been identified that bind regions of HuR known to be essential for HuR's ability to shuttle between the nucleus and the cytoplasm and to stabilize mRNA: SETalpha, SETbeta, pp32, and acidic protein rich in leucine (APRIL). Three have been reported to be protein phosphatase 2A inhibitors. All four ligands contain long, acidic COOH-terminal tails, while pp32 and APRIL share a second motif: rev-like leucine-rich repeats in their NH(2)-terminal regions. pp32 and APRIL are nucleocytoplasmic shuttling proteins that interact with the nuclear export factor CRM1 (chromosomal region maintenance protein 1). The inhibition of CRM1 by leptomycin B leads to the nuclear retention of pp32 and APRIL, their increased association with HuR, and an increase in HuR's association with nuclear poly(A)+ RNA. Furthermore, transcripts from the ARE-containing c-fos gene are selectively retained in the nucleus, while the cytoplasmic distribution of total poly(A)+ RNA is not altered. These data provide evidence that interaction of its ligands with HuR modulates HuR's ability to bind its target mRNAs in vivo and suggest that CRM1 is instrumental in the export of at least some cellular mRNAs under certain conditions. Discussed is the possible role of these ligands upstream of HuR in pathways that govern the stability of ARE-containing mRNAs (Brennan, 2000).

The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro that correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. HuR was transiently transfected into mouse L929 cells: overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, it was demonstrated that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment (Fan, 1998a).

ELAV-like proteins and learning

The view that memory is encoded by variations in the strength of synapses implies that long-term biochemical changes take place within subcellular microdomains of neurons. These changes are thought ultimately to be an effect of transcriptional regulation of specific genes. Localized changes, however, cannot be fully explained by a purely transcriptional control of gene expression. The neuron-specific ELAV-like HuB, HuC, and HuD RNA-binding proteins act posttranscriptionally by binding to adenine- and uridine-rich elements (AREs) in the 3' untranslated region of a set of target mRNAs, and by increasing mRNA cytoplasmic stability and/or rate of translation. Neuronal ELAV-like genes undergo a sustained up-regulation in hippocampal pyramidal cells only of mice and rats that have learned a spatial discrimination paradigm. This learning-specific increase of ELAV-like proteins is localized within cytoplasmic compartments of the somata and proximal dendrites and is associated with the cytoskeleton. This increase is also accompanied by enhanced expression of the GAP-43 gene, known to be regulated mainly posttranscriptionally and whose mRNA is demonstrated here to be an in vivo ELAV-like target. Antisense-mediated knockdown of HuC impairs spatial learning performance in mice and induces a concomitant down-regulation of GAP-43 expression. Neuronal ELAV-like proteins could exert learning-induced posttranscriptional control of an array of target genes uniquely suited to subserve substrates of memory storage (Quattrone, 2001).

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elav: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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