Ann Bin Shyu

The p38 MAP kinase pathway signals for cytokine- induced mRNA stabilization via MAP kinase- activated protein kinase 2 and an AU-rich region- targeted mechanism

Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline‐controlled expression system to determine the half‐lives of interleukin (IL)‐6 and IL‐8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal‐induced stabilization was transferred to the stable β‐globin mRNA by a 161‐nucleotide fragment of IL‐8 mRNA which contains an AU‐rich region, as well as by defined AU‐rich elements (AREs) of the c‐fos and GM‐CSF mRNAs. Of the different MEKK1‐activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF‐κB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant‐negative mutant of p38 MAP kinase interfered with MEKK1 and also IL‐1‐induced stabilization. Furthermore, an active form of the p38 MAP kinase‐activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6‐induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress‐induced gene expression by stabilizing mRNAs through an MK2‐dependent, ARE‐targeted mechanism.

RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein

An important paradigm for post‐transcriptional regulation is the control of cytoplasmic mRNA stability mediated by AU‐rich elements (AREs) in the 3′ untranslated region of transcripts encoding oncoproteins, cytokines and transcription factors. While many RNA‐binding proteins have been shown to bind to AREs in vitro, neither the functional consequences nor the physiological significance of their interactions are known. Here we demonstrate a role for the embryonic lethal abnormal visual (ELAV) RNA‐binding protein HuR in mRNA turnover in vivo. The ELAV family of RNA‐binding proteins is highly conserved in vertebrates. In humans, there are four members; HuR is expressed in all proliferating cells, whereas Hel‐N1, HuC and HuD are expressed in terminally differentiated neurons. We show that elevation of cytoplasmic HuR levels inhibits c‐fos ARE‐mediated RNA decay but has little effect on rapid decay directed by c‐jun ARE. It appears that HuR has little effect on deadenylation but delays onset of decay of the RNA body and slows down its subsequent decay. We also show that HuR can be induced to redistribute from the nucleus to the cytoplasm and that this redistribution is associated with an altered function. Modulation of the ARE‐mediated decay pathway through controlling distribution of the ELAV proteins between nucleus and cytoplasm may be a mechanism by which cell growth and differentiation is regulated.

Messenger RNA regulation: to translate or to degrade

Quality control of gene expression operates post‐transcriptionally at various levels in eukaryotes. Once transcribed, mRNAs associate with a host of proteins throughout their lifetime. These mRNA–protein complexes (mRNPs) undergo a series of remodeling events that are influenced by and/or influence the translation and mRNA decay machinery. In this review we discuss how a decision to translate or to degrade a cytoplasmic mRNA is reached. Nonsense‐mediated mRNA decay (NMD) and microRNA (miRNA)‐mediated mRNA silencing are provided as examples. NMD is a surveillance mechanism that detects and eliminates aberrant mRNAs whose expression would result in truncated proteins that are often deleterious to the organism. miRNA‐mediated mRNA silencing is a mechanism that ensures a given protein is expressed at a proper level to permit normal cellular function. While NMD and miRNA‐mediated mRNA silencing use different decision‐making processes to determine the fate of their targets, both are greatly influenced by mRNP dynamics. In addition, both are linked to RNA processing bodies. Possible modes involving 3′ untranslated region and its associated factors, which appear to play key roles in both processes, are discussed.

Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover

In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon–containing unstable β-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.

A Mechanism for Translationally Coupled mRNA Turnover: Interaction between the Poly(A) Tail and a c-fos RNA Coding Determinant via a Protein Complex

mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.

The Double Lives of Shuttling mRNA Binding Proteins

Several other RNA binding proteins besides hnRNPs have been identified that not only function only in the nucleus but also regulate events in the cytoplasm. These include the transcription factors Bicoid and FRGY2, and the RNA splicing regulator SXL, which have all recently been shown to regulate translation (7xGray, N.K and Wickens, M. Annu. Rev. Cell Dev. Biol. 1998; 14: 399–458Crossref | PubMed | Scopus (412)See all References, 17xMatsumoto, K, Wassarman, K, and Wolffe, A.P. EMBO J. 1998; 17: 2107–2121Crossref | PubMed | Scopus (151)See all References and references therein). Our knowledge of these RNA binding proteins suggests that many of them first complex with pre-mRNAs in the nucleus to regulate their activities and then escort mature mRNAs out to the cytoplasm to further influence their behavior. However, this appealing scenario remains unproven. Even in the case of proteins known to shuttle between the nucleus and the cytoplasm, it is not known whether these RNA binding proteins actually direct (rather than follow) mRNAs to the cytoplasm. In fact, it is not even clear whether most shuttling RNA binding proteins are bound to RNA when they traverse the nuclear pore. Instead, many RNA binding proteins may travel to the cytoplasm alone. Another unanswered question is how shuttling RNA binding proteins reach the cytoplasm. Do most of them emigrate from the nucleus, or does a selected subset set up permanent residence at their place of origin in the cytoplasm?Despite these unanswered questions, the observation that many shuttling RNA binding proteins perform duties in both the nucleus and the cytoplasm suggests the following model (Figure 1Figure 1). In the nucleus, shuttling proteins promote the assembly of a proper mRNP (mRNA/protein) complex, permitting its export to the cytoplasm. After emerging from the nucleus, these RNA binding proteins then direct the assembly or reorganization of the RNP complex to help dictate the specific outcome appropriate for a given mRNA. According to this model, the particular shuttling proteins bound to the mRNA determines where the mRNA will be localized in the cytoplasm, its rate of translation, and its rate of decay. It is possible that these shuttling proteins regulate these posttranscriptional events by interacting with microtubules, ribosomes, and degradosomes, either directly or via adaptor proteins.Figure 1Model for How Shuttling mRNA Binding Proteins Regulate Events in Both the Nucleus and the CytoplasmPABP, poly(A) binding protein; eIF4F, eukaryotic initiation factor 4F.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe notion that nuclear proteins travel with mRNAs to the cytoplasm to regulate mRNA function provides an explanation for a series of observations that the nuclear history of an mRNA can affect its cytoplasmic fate. For example, some genes have been shown to require introns for efficient translation of their spliced mRNA products in the cytoplasm (Matsumoto et al. 1998xMatsumoto, K, Wassarman, K, and Wolffe, A.P. EMBO J. 1998; 17: 2107–2121Crossref | PubMed | Scopus (151)See all ReferencesMatsumoto et al. 1998). In addition, as mentioned earlier, introns are necessary to engage the mammalian NMD RNA surveillance pathway that detects nonsense codons by a mechanism with features of the cytoplasmic translational machinery. To explain these observations, it has been hypothesized that RNA binding proteins exist that regulate intron-dependent events in the nucleus and then go on to the cytoplasm to regulate subsequent events. Future studies will be required to test this theory and, if it is true, to identify the specific RNA binding shuttling proteins involved and how they communicate with each other.How RNA binding shuttling proteins themselves are regulated also remains to be determined. Is their distribution between the nucleus and the cytoplasm modulated by environmental cues? Do posttranslational events, such as phosphorylation and methylation, alter their ability to control gene expression events in the nucleus or the cytoplasm? The answer appears to be yes, as a recent study showed that the p38 stress-activated mitogen-activated kinase alters the nucleocytoplasmic distribution of hnRNP A1 in response to osmotic stress, leading to changes of alternative splicing (van der Houven van Oordt et al. 2000xvan der Houven van Oordt, W, Diaz-Meco, M.T, Lozano, J, Krainer, A.R, Moscat, J, and Caceres, J.F. J. Cell Biol. 2000; 149: 307–316Crossref | PubMed | Scopus (224)See all Referencesvan der Houven van Oordt et al. 2000). As stress-activated signaling pathways are fundamental to the life and death of cells, they could play a key role in modulating the functional linkage between mRNA metabolism in the cytoplasmic and nuclear compartments under both physiological conditions and stress responses. The answers to these questions will ultimately tell us much about how communication between the nuclear and cytoplasmic compartments is orchestrated.‡To whom correspondence should be addressed (e-mail: ann-bin.shyu@uth.tmc.edu).

Versatile Role for hnRNP D Isoforms in the Differential Regulation of Cytoplasmic mRNA Turnover

ABSTRACT An important emerging theme is that heterogeneous nuclear ribonucleoproteins (hnRNPs) not only function in the nucleus but also control the fates of mRNAs in the cytoplasm. Here, we show that hnRNP D plays a versatile role in cytoplasmic mRNA turnover by functioning as a negative regulator in an isoform-specific and cell-type-dependent manner. We found that hnRNP D discriminates among the three classes of AU-rich elements (AREs), most effectively blocking rapid decay directed by class II AREs found in mRNAs encoding cytokines. Our experiments identified the overlapping AUUUA motifs, one critical characteristic of class II AREs, to be the key feature recognized in vivo by hnRNP D for its negative effect on ARE-mediated mRNA decay. The four hnRNP D isoforms, while differing in their ability to block decay of ARE-containing mRNAs, all potently inhibited mRNA decay directed by another mRNA cis element that shares no sequence similarity with AREs, the purine-rich c-fosprotein-coding region determinant of instability. Further experiments indicated that different mechanisms underlie the inhibitory effect of hnRNP D on the two distinct mRNA decay pathways. Our study identifies a potential mechanism by which cytoplasmic mRNA turnover can be differentially and selectively regulated by hnRNP D isoforms in mammalian cells. Our results support the notion that hnRNP D serves as a key factor broadly involved in general mRNA decay.

Highly Selective Actions of HuR in Antagonizing AU-Rich Element-Mediated mRNA Destabilization

ABSTRACT Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5′ AUUUA-containing domain and the 3′ U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Ago–TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2–Pan3 and Ccr4–Caf1 deadenylase complexes followed by Dcp1–Dcp2 complex–directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.