Mitochondrial poly(A) polymerase and polyadenylation

Polyadenylation of mitochondrial RNAs in higher eukaryotic organisms have diverse effects on their function and metabolism. Polyadenylation completes the UAA stop codon of a majority of mitochondrial mRNAs in mammals, regulates the translation of the mRNAs, and has diverse effects on their stability. In contrast, polyadenylation of most mitochondrial mRNAs in plants leads to their degradation, consistent with the bacterial origin of this organelle. PAPD1 (mtPAP, TUTase1), a noncanonical poly(A) polymerase (ncPAP), is responsible for producing the poly(A) tails in mammalian mitochondria. The crystal structure of human PAPD1 was reported recently, offering molecular insights into its catalysis. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail

The CCCH family of tandem zinc finger proteins has recently been shown to promote the turnover of certain mRNAs containing class II AU-rich elements (AREs). In the case of one member of this family, tristetraprolin (TTP), absence of the protein in knockout mice leads to stabilization of two mRNAs containing AREs of this type, those encoding tumor necrosis factor alpha (TNFalpha) and granulocyte-macrophage colony-stimulating factor. To begin to decipher the mechanism by which these zinc finger proteins stimulate the breakdown of this class of mRNAs, we co-transfected TTP and its related CCCH proteins into 293 cells with vectors encoding full-length TNFalpha, granulocyte-macrophage colony-stimulating factor, and interleukin-3 mRNAs. Co-expression of the CCCH proteins caused the rapid turnover of these ARE-containing mRNAs and also promoted the accumulation of stable breakdown intermediates that were truncated at the 3'-end of the mRNA, even further 5' than the 5'-end of the poly(A) tail. To determine whether an intact poly(A) tail was necessary for TTP to promote this type of mRNA degradation, we inserted the TNFalpha ARE into a nonpolyadenylated histone mRNA and also attached a histone 3'-end-processing sequence to the 3'-end of nonpolyadenylated interleukin-3 and TNFalpha mRNAs. In all three cases, TTP stimulated the turnover of the ARE-containing mRNAs, despite the demonstrated absence of a poly(A) tail. These studies indicate that members of this class of CCCH proteins can promote class II ARE-containing mRNA turnover even in the absence of a poly(A) tail, suggesting that the processive removal of the poly(A) tail may not be required for this type of CCCH protein-stimulated mRNA turnover.     

Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs.

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.     

Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.     

Mitochondrial Dysfunction Reveals the Role of mRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular Dystrophy Pathogenesis

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.     

The poly(A)-poly(A)-binding protein complex is a major determinant of mRNA stability in vitro.

Using an in vitro mRNA decay system, we investigated how poly(A) and its associated poly(A)-binding protein (PABP) affect mRNA stability. Cell extracts used in the decay reactions were depleted of functional PABP either by adding excess poly(A) competitor or by passing the extracts over a poly(A)-Sepharose column. Polyadenylated mRNAs for beta-globin, chloramphenicol acetyltransferase, and simian virus 40 virion proteins were degraded 3 to 10 times faster in reactions lacking PABP than in those containing excess PABP. The addition of purified Saccharomyces cerevisiae or human cytoplasmic PABP to PABP-depleted reactions stabilized the polyadenylated mRNAs. In contrast, the decay rates of nonpolyadenylated mRNAs were unaffected by PABP, indicating that both the poly(A) and its binding protein were required for maintaining mRNA stability. A nonspecific single-stranded binding protein from Escherichia coli did not restore stability to polyadenylated mRNA, and the stabilizing effect of PABP was inhibited by anti-PABP antibody. The poly(A) tract was the first mRNA segment to be degraded in PABP-depleted reactions, confirming that the poly(A)-PABP complex was protecting the 3' region from nucleolytic attack. These results indicate that an important function of poly(A), in conjunction with its binding protein, is to protect polyadenylated mRNAs from indiscriminate destruction by cellular nucleases. A model is proposed to explain how the stability of an mRNA could be affected by the stability of its poly(A)-PABP complex.     

Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.     

Transcriptional analysis of estrogen receptor alpha variant mRNAs in colorectal cancers and their matched normal colorectal tissues

Estrogen is involved in suppression of colorectal cancer development and exerts its function via estrogen receptors alpha, beta and their splicing variants. Whether the recently indentified ER-alpha splicing variants, ER-alpha36 and ER-alpha46, play a role in colorectal cancer development is unknown. In this study, we quantified the mRNA copy numbers of wild type ER-alpha (ER-alpha66), ER-alpha46 and ER-alpha36 in 35 colorectal cancers and their matched normal colorectal tissues by quantitative real-time PCR assay, and correlated their mRNA levels with the clinicopathological properties of the tumors. We found that ER-alpha66, ER-alpha46 and ER-alpha36 mRNAs were coexpressed in all colorectal cancers and their matched normal tissues. The decreased mRNA levels of ER-alpha36 and ER-alpha46 whereas no difference of ER-alpha66 mRNA was observed in colorectal cancers compared to their matched normal tissues. Moreover, change in the expression of ER-alpha36 mRNA level was correlated with Dukes' stage of the tumor and the lymph node metastasis. ER-alpha36 mRNA was decreased significantly in Dukes' C+D compared to Dukes' A+B stage tumors (P=0.017), and the expression of ER-alpha36 mRNA in N(1)/N(2) was lower than that in N(0) lymph node metastasis (P=0.049). So ER-alpha36 and ER-alpha46 might be implicated in the development and progression of colorectal cancers.     

ALKBH5 Promotes Multiple Myeloma Tumorigenicity through inducing m6A-demethylation of SAV1 mRNA and Myeloma Stem Cell Phenotype

N6-methyladenosine (m⁶A) is the most prevalent modification to RNA in higher eukaryotes. ALKBH5 is an RNA demethylase that impacts RNA export and metabolism, and its aberrant expression is associated with the generation of tumours. In this study, we found that ALKBH5 was highly expressed in both primary CD138⁺ plasma cells isolated from multiple myeloma (MM) patients and MM cell lines. Downregulation of ALKBH5 inhibited myeloma cell proliferation, neovascularization, invasion and migration ability, and promoted the apoptosis in vivo and in vitro. MeRIP-seq identified the SAV1 gene as main target gene of ALKBH5. Inhibiting ALKBH5 in MM cells increased SAV1 m⁶A levels, decreased SAV1 mRNA stability and expression, suppressed the stem cell related HIPPO-pathway signalling and ultimately activates the downstream effector YAP, exerting an anti-myeloma effect. Additionally, MM stem cell phenotype was suppressed in ALKBH5-deficient cells and the expression of pluripotency factors NANOG, SOX2 and OCT4 were also decreased. Altogether, our results suggest that ALKBH5 acts as an oncogene in MM and might serve as an attractive potential biomarker and therapeutic target.     

Poly(A) tail length-dependent stabilization of GAP-43 mRNA by the RNA-binding protein HuD

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. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-based in vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was 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, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude 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.     

The differential distribution of beta tubulin mRNAs in individual mammalian brain cells

It has been shown by in vitro translation of polyadenylated messenger RNAs (poly(A)+ mRNAs) that the mRNAs encoding both alpha and beta tubulin isotypes are present at much higher relative levels in the developing rat brain than they are in the adult, suggesting that the requirements for tubulin subunits vary with cell type and/or with the developmental stages of a particular cell type. The postnatally developing rat cerebellum, with its readily identifiable cell populations that perform the gamut of developmental tasks, is a suitable model for analyzing specific cellular mRNA distributions during development. In this report, by in situ hybridization techniques it is shown that, by comparison to total cellular poly(A)+ mRNA levels, there is relatively more of the total beta tubulin mRNAs in mitotically active external granule layer cells than in those in the internal granule layer. These results show that migration and differentiation of these granule cells is accompanied by a decrease in their beta tubulin mRNA levels relative to the levels in granule cells of the external granule cell layer. Furthermore, the relative levels of beta tubulin mRNA both in the prenatally formed Purkinje cells and the postnatally formed stellate cells are two to fourfold less than in the granule cells of the internal granule cell layer.     

Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5''-terminal poly(A) sequences.

We have identified and characterized one of the most strongly-expressed genes of cowpox virus (CPV). This is the gene encoding the major protein component of the A-type inclusion bodies produced by this virus. This gene (designated the 160K gene) is transcribed late during the infection. Analyses of its mRNAs showed that these late RNAs, unlike all other characterized late mRNAs of poxviruses, are uniform in length. However, the most remarkable feature of the mRNAs of the 160K gene is the structure of their 5'-termini. Most of these mRNAs have 5'-terminal poly(A) sequences containing 5-21 residues. Furthermore, these 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. Instead, the nucleotide sequences of the mRNA and the viral DNA diverge at the site of the three As in the sequence 5'-TAAATG-3' containing the gene's initiation codon. Consequently, the poly(A) provides the leader sequences of these mRNAs. These unusual 5'-terminal structures suggest that the late mRNAs of pox-virus genes are generated by a novel process.     

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.     

Expression of a micro-protein.

The smallest known open reading frame encodes the ribosomal protein L41, which in yeast is composed of only 24 amino acids, 17 of which are arginine or lysine. Because of the unique problems that might attend the translation of such a short open reading frame, we have investigated the properties and the translation of the mRNAs encoding L41. In Saccharomyces cerevisiae L41 is encoded by two linked genes, RPL41A and RPL41B. These genes give rise to mRNAs that have short 5' leaders of 18 and 22 nucleotides and rather long 3' leaders of 203 and 210 nucleotides not including their poly(A) tails. The mRNAs are translated exclusively on monosomes, suggesting that ribosomes do not remain attached to the mRNA after termination of translation. Calculations based on the abundance of ribosomes and of L41 mRNA indicate that the entire translation event, from initiation through termination, must occur in approximately 2 s. Termination of translation after only 25 codons does not subject the mRNAs encoding L41 to nonsense-mediated decay. Surprisingly, despite the L41 ribosomal protein being conserved from the archaea through the mammalia, S. cerevisiae can grow relatively normally after deletion of both RPL41A and RPL41B.     

Stress mRNA metabolism in canavanine-treated chicken embryo cells.

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.     

Yeast Gis2 and Its Human Ortholog CNBP Are Novel Components of Stress-Induced RNP Granules

Although a CCTG expansion in the gene encoding the zinc knuckle protein CNBP causes a common form of muscular dystrophy, the function of both human CNBP and its putative budding yeast ortholog Gis2 remain poorly understood. Here we report the protein interactions of Gis2 and the subcellular locations of both Gis2 and CNBP. We found that Gis2 exhibits RNA-dependent interactions with two proteins involved in mRNA recognition, the poly(A) binding protein and the translation initiation factor eIF4G. We show that Gis2 is a component of two large RNA-protein granules, processing bodies and stress granules, which contain translationally repressed mRNAs. Consistent with a functional ortholog, CNBP also associates with the poly(A) binding protein and accumulates in stress granules during arsenite treatment of human cells. These results implicate both Gis2 and CNBP in mRNA handling during stress.