Functional mapping of destabilizing elements in the protein-coding region of the Drosophila fushi tarazu mRNA

The instability of the fushi tarazu (ftz) mRNA is essential for the proper development of the Drosophila embryo. Previously, we identified a 201-nucleotide instability element (FIE3) in the 3' untranslated region (UTR) of the ftz mRNA. Here we report on the identification of two additional elements in the protein-coding region of the message: the 63-nucleotide-long FIE5-1 and the 69-nucleotide-long FIE5-2. The function of both elements was position-dependent; the same elements destabilized RNAs when present within the coding region but did not when embedded in the 3' UTR of the hybrid mRNAs. We conclude that ftz mRNA has three redundant instability elements, two in the protein-coding region and one in the 3' UTR. Although each instability element is sufficient to destabilize a heterologous mRNA, the destabilizing activity of the two 5'-elements depended on their position within the message.     

Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos

During ascidian embryogenesis, some mRNAs show clear localization at the posterior-most region. These postplasmic mRNAs are divided into two groups (type I and type II) according to their pattern of localization. To elucidate how these localization patterns are achieved, we attempted to identify the localization elements of these mRNAs. When in vitro synthesized postplasmic mRNAs were introduced into eggs, these mRNAs showed posterior localization similar to the endogenous mRNAs. The posterior localization of these mRNAs was mediated by their 3' untranslated regions (3' UTRs), as is the case for several localized Drosophila and Xenopus mRNAs. We identified smaller fragments of the 3' UTRs of HrWnt-5 and HrPOPK-1 mRNAs (type I) and HrPet-3 mRNA (type II) that were sufficient to direct green fluorescent protein mRNA to the posterior pole. For the localization of HrWnt-5 mRNA, two UG dinucleotide repetitive elements were essential. Motifs similar to these small elements also exist within the HrPOPK-1 mRNA localization element and 3' UTR of HrZF-1 mRNA, suggesting the conservation of localization elements among type I mRNAs. In contrast, the smallest sequence that suffices for the posterior localization of HrPet-3 (a type II mRNA) has different features from those of type I mRNAs; indeed, it does not have an identifiable critical element. This difference may distinguish type II mRNAs from type I mRNAs. These findings, especially the identification of the small localization element of HrWnt-5 mRNA, provide new insights into the localization of mRNAs during ascidian embryogenesis.     

p190RhoGEF Binds to a destabilizing element in the 3' untranslated region of light neurofilament subunit mRNA and alters the stability of the transcript.

Stabilization of neurofilament (NF) mRNAs plays a major role in regulating levels of NF expression and in establishing axonal size and rate of axonal conduction. Previous studies have identified a 68-nucleotide destabilizing element at the junction of the coding region and 3' untranslated region of the light NF subunit (NF-L) mRNA. The present study has used the destabilizing element (probe A) to screen a rat brain cDNA library for interactive proteins. A cDNA clone encoding 1068 nucleotides in the C-terminal domain of p190RhoGEF (clone 39) was found to bind strongly and specifically to the RNA probe. The interaction was confirmed using a glutathione S-transferase/clone 39 fusion protein in Northwestern, gel-shift, and cross-linkage studies. The glutathione S-transferase/clone 39 fusion protein also enhanced the cross-linkage of a major 43-kDa protein in brain extract to the destabilizing element. Functional studies on stably transfected neuronal cells showed that p190RhoGEF expression increased the half-life of a wild-type NF-L mRNA but did not alter the half-life of a mutant NF-L mRNA lacking the destabilizing element. The findings reveal a novel interactive feature of p190RhoGEF that links the exchange factor with NF mRNA stability and regulation of the axonal cytoskeleton.     

Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.     

Messenger RNA degradation in Saccharomyces cerevisiae

The analysis of 17 functional mRNAs and two recombinant mRNAs in the yeast Saccharomyces cerevisiae suggests that the length of an mRNA influences its half-life in this organism. The mRNAs are clearly divisible into two populations when their lengths and half-lives are compared. Differences in ribosome loading amongst the mRNAs cannot account for this division into relatively stable and unstable populations. Also, specific mRNAs seem to be destabilized to differing extents when their translation is disrupted by N-terminus-proximal stop codons. The analysis of a mutant mRNA, generated by the fusion of the yeast PYK1 and URA3 genes, suggests that a destabilizing element exists within the URA3 sequence. The presence of such elements within relatively unstable mRNAs might account for the division between the yeast mRNA populations. On the basis of these, and other previously published observations, a model is proposed for a general pathway of mRNA degradation in yeast. This model may be relevant to other eukaryotic systems. Also, only a minor extension to the model is required to explain how the stability of some eukaryotic mRNAs might be regulated.     

Hypoxic regulation of vascular endothelial growth factor mRNA stability requires the cooperation of multiple RNA elements

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3' untranslated region (3'UTR), but also contains destabilizing elements in the 5'UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5'UTR, coding region, and 3'UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.     

Tumor necrosis factor alpha-induced glucose transporter (GLUT-1) mRNA stabilization in 3T3-L1 preadipocytes. Regulation by the adenosine-uridine binding factor.

Tumor necrosis factor alpha (TNF alpha), 12-O-tetradecanoylphorbol-13-acetate and cAMP stimulate hexose transport in quiescent 3T3-L1 preadipocytes by stabilizing the relatively labile mRNA coding for the basal glucose transporter, GLUT-1. The 3'-UTR of GLUT-1 mRNA contains a single copy of the destabilizing AUUUA motif in the context of an AU-rich region. The adenosine-uridine binding factor (AUBF) is a cytosolic protein which interacts with similar AU-rich regions in a variety of labile cytokine and oncogene mRNAs. Here, we demonstrate that AUBF complexes in vitro with GLUT-1 mRNA through the AU-rich portion of the 3'-UTR. AUBF activity is very low in quiescent preadipocytes, but can be up-regulated by agonists such as TPA, TNF alpha, cAMP, and okadaic acid, all of which stabilize GLUT-1 mRNA. The time courses of TNF alpha- and TPA-mediated AUBF up-regulation and GLUT-1 mRNA stabilization are coincident, suggesting a cause and effect relationship.     

Can ID repetitive elements serve as cis-acting dendritic targeting elements? An in vivo study

Dendritic localization of mRNA/RNA involves interaction of cis-elements and trans-factors. Small, non-protein coding dendritic BC1 RNA is thought to regulate translation in dendritic microdomains. Following microinjections into cultured cells, BC1 RNA fused to larger mRNAs appeared to impart transport competence to these chimeras, and its 5' ID region was proposed as the cis-acting dendritic targeting element. As these ID elements move around rodent genomes and, if transcribed, form a long RNA stem-loop, they might, thereby, lead to new localizations for targeted gene products. To test their targeting ability in vivo we created transgenic mice expressing various ID elements fused to the 3' UTR of reporter mRNA for Enhanced Green Fluorescent Protein. In vivo, neither ID elements nor the BC1 RNA coding region were capable of transporting EGFP RNA to dendrites, although the 3' UTR of alpha-CaMKII mRNA, an established cis-acting element did produce positive results. Other mRNAs containing naturally inserted ID elements are also not found in neuronal dendrites. We conclude that the 5' ID domain from BC1 RNA is not a sufficient dendritic targeting element for mRNAs in vivo.     

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

Control of translation by the 5'- and 3'-terminal regions of the dengue virus genome

The genomic RNAs of flaviviruses such as dengue virus (DEN) have a 5' m7GpppN cap like those of cellular mRNAs but lack a 3' poly(A) tail. We have studied the contributions to translational expression of 5'- and 3'-terminal regions of the DEN serotype 2 genome by using luciferase reporter mRNAs transfected into Vero cells. DCLD RNA contained the entire DEN 5' and 3' untranslated regions (UTRs), as well as the first 36 codons of the capsid coding region fused to the luciferase reporter gene. Capped DCLD RNA was as efficiently translated in Vero cells as capped GLGpA RNA, a reporter with UTRs from the highly expressed alpha-globin mRNA and a 72-residue poly(A) tail. Analogous reporter RNAs with regulatory sequences from West Nile and Sindbis viruses were also strongly expressed. Although capped DCLD RNA was expressed much more efficiently than its uncapped form, uncapped DCLD RNA was translated 6 to 12 times more efficiently than uncapped RNAs with UTRs from globin mRNA. The 5' cap and DEN 3' UTR were the main sources of the translational efficiency of DCLD RNA, and they acted synergistically in enhancing translation. The DEN 3' UTR increased mRNA stability, although this effect was considerably weaker than the enhancement of translational efficiency. The DEN 3' UTR thus has translational regulatory properties similar to those of a poly(A) tail. Its translation-enhancing effect was observed for RNAs with globin or DEN 5' sequences, indicating no codependency between viral 5' and 3' sequences. Deletion studies showed that translational enhancement provided by the DEN 3' UTR is attributable to the cumulative contributions of several conserved elements, as well as a nonconserved domain adjacent to the stop codon. One of the conserved elements was the conserved sequence (CS) CS1 that is complementary to cCS1 present in the 5' end of the DEN polyprotein open reading frame. Complementarity between CS1 and cCS1 was not required for efficient translation.     

Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element.

SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons. In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region. Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function. These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs. Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously. We also define base pairing requirements in the stem of this element. In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function. Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.     

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.