An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay

The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting factors, including Barentsz and the Mago-Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago-Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago-Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)-Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.     

Induction of germ cell formation by oskar.

The oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three, oskar, vasa and tudor, are essential at an ectopic site.     

Cleavage of 5' splice site and lariat formation are independent of 3' splice site in yeast mRNA splicing

Analysis of messenger RNA splicing in yeast and in metazoa has led to the identification of an RNA molecule in a lariat conformation. This structure has been found as an mRNA splicing intermediate in vitro and identical molecules have been identified in vivo. Lariat formation involves cleavage of the precursor at the 5' splice site (5' SS) and the formation of a 2'-5' phosphodiester bond between the guanosine residue at the 5' end of the intron and an adenosine within the intron. The yeast branchpoint is located within the absolutely conserved TACTAAC box (that is, the last A of the TACTAAC box is the site of formation of the 2'-5' phosphodiester bond with the 5' end of the intron)3,4. Moreover, efficient 5' SS cleavage and lariat formation require proper sequences at the 5' splice junction and within the TACTAAC box. Here we demonstrate that 5' SS cleavage and lariat formation take place in vitro in the absence of the 3' SS and much of the 3' junction. These results are discussed in light of possible differences between yeast and metazoan mRNA splicing.     

The Drosophila gene torso encodes a putative receptor tyrosine kinase

The maternal gene torso, required for determination of anterior and posterior terminal structures in the Drosophila embryo, was cloned using P-element tagging. Genetic evidence suggests that the action of the gene product is spatially restricted to the terminal regions; the torso messenger RNA, however, is evenly distributed. Structural similarities of the predicted torso protein with growth-factor receptor tyrosine kinases suggest that the spatial restriction of torso activity results from a localized activation of the torso protein at the anterior and posterior egg pole.     

bicoid RNA localization requires specific binding of an endosomal sorting complex

bicoid messenger RNA localizes to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first localized cytoplasmic determinant to be identified, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that the mRNA is recognized by a complex of many redundant proteins, each of which binds to the localization element in the 3' untranslated region (UTR) with little or no specificity. Indeed, the only known RNA-binding protein that co-localizes with bicoid mRNA is Staufen, which binds non-specifically to double-stranded RNA in vitro. Here we show that mutants in all subunits of the ESCRT-II complex (VPS22, VPS25 and VPS36) abolish the final Staufen-dependent step in bicoid mRNA localization. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm. However, the role of ESCRT-II in bicoid localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and III components do not affect the targeting of bicoid mRNA. Instead, VPS36 functions by binding directly and specifically to stem-loop V of the bicoid 3' UTR through its amino-terminal GLUE domain, making it the first example of a sequence-specific RNA-binding protein that recognizes the bicoid localization signal. Furthermore, VPS36 localizes to the anterior of the oocyte in a bicoid-mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This function of ESCRT-II as an RNA-binding complex is conserved in vertebrates and may clarify some of its roles that are independent of endosomal sorting.     

Type I iodothyronine deiodinase is a selenocysteine-containing enzyme.

Although thyroxine (3,5,3',5'-tetraiodothyronine, T4) is the principal secretory product of the vertebrate thyroid, its essential metabolic and developmental effects are all mediated by 3,5,3'-triiodothyronine (T3), which is produced from the prohormone by 5'-deiodination. The type-I iodothyronine deiodinase, a thiol-requiring propylthiouracil-sensitive oxidoreductase, is found mainly in liver and kidney and provides most of the circulating T3(1) but so far this enzyme has not been purified. Using expression cloning in the Xenopus oocyte, we have isolated a 2.1-kilobase complementary DNA for this deiodinase from a rat liver cDNA library. The kinetic properties of the protein expressed in transient assay systems, the tissue distribution of the messenger RNA, and its changes with thyroid status, all confirm its identity. We find that the mRNA for this enzyme contains a UGA codon for selenocysteine which is necessary for maximal enzyme activity. This explains why conversion of T4 to T3 is impaired in experimental selenium deficiency and identifies an essential role for this trace element in thyroid hormone action.     

The ribosome uses two active mechanisms to unwind messenger RNA during translation

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.     

The human RNA kinase hClp1 is active on 3' transfer RNA exons and short interfering RNAs

RNA interference allows the analysis of gene function by introducing synthetic, short interfering RNAs (siRNAs) into cells. In contrast to siRNA and microRNA duplexes generated endogenously by the RNaseIII endonuclease Dicer, synthetic siRNAs display a 5' OH group. However, to become incorporated into the RNA-induced silencing complex (RISC) and mediate target RNA cleavage, the guide strand of an siRNA needs to display a phosphate group at the 5' end. The identity of the responsible kinase has so far remained elusive. Monitoring siRNA phosphorylation, we applied a chromatographic approach that resulted in the identification of the protein hClp1 (human Clp1), a known component of both transfer RNA splicing and messenger RNA 3'-end formation machineries. Here we report that the kinase hClp1 phosphorylates and licenses synthetic siRNAs to become assembled into RISC for subsequent target RNA cleavage. More importantly, we reveal the physiological role of hClp1 as the RNA kinase that phosphorylates the 5' end of the 3' exon during human tRNA splicing, allowing the subsequent ligation of both exon halves by an unknown tRNA ligase. The investigation of this novel enzymatic activity of hClp1 in the context of mRNA 3'-end formation, where no RNA phosphorylation event has hitherto been predicted, remains a challenge for the future.     

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.     

Drosophila nurse cells produce a posterior signal required for embryonic segmentation and polarity

The segmental pattern of insect embryos depends on influences from morphogenetic centres near each of the egg poles. In Drosophila, maternal effect mutations are known that impair the normal function of each centre. Injection of wild-type cytoplasm into mutant eggs has revealed that morphogenetic signals localized at the anterior and posterior pole of eggs can be transplanted. We show here that these activities can also be detected during oogenesis. Posterior activity can be recovered at an early stage (stage 10, ref. 5) from the oocyte-nurse cell complex, but anterior activity can only be detected in the mature oocytes (stage 14). We conclude that the bicoid-dependent anterior signal, although produced by the nurse cells, does not become active before it is localized to the anterior egg pole, whereas posterior activity can be detected in the nurse cells before, and therefore independently of, its localization to the posterior egg pole.     

Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes

A prominent hypothesis in embryology is that localized maternal factors are important in specifying cell fate. There are, however, only a few examples of maternal molecules that have been shown to be localized and very little is known about how such factors are physically localized within an egg (for review see ref. 1). Previously, cDNA clones were obtained for a class of localized maternal mRNAs from Xenopus laevis. These mRNAs are unusual in that they are concentrated at either the animal or vegetal pole of unfertilized eggs. In the present study the synthesis and intracellular distribution of one of them, Vg1, has been examined during oogenesis. The results show that Vg1 mRNA is localized as a crescent at the vegetal pole of mature oocytes. Surprisingly, this mRNA is uniformly distributed in the cytoplasm of immature oocytes. These findings suggest that a single cell, the frog oocyte, has some mechanism for translocating specific RNAs like Vg1. The process that moves Vg1 mRNA is evidently a cytoplasmic localization machinery which is not directly coupled to the synthesis of Vg1 RNA.     

Yeast Sm-like proteins function in mRNA decapping and decay

One of the main mechanisms of messenger RNA degradation in eukaryotes occurs by deadenylation-dependent decapping which leads to 5'-to-3' decay. A family of Sm-like (Lsm) proteins has been identified, members of which contain the 'Sm' sequence motif, form a complex with U6 small nuclear RNA and are required for pre-mRNA splicing. Here we show that mutations in seven yeast Lsm proteins (Lsm1-Lsm7) also lead to inhibition of mRNA decapping. In addition, the Lsm1-Lsm7 proteins co-immunoprecipitate with the mRNA decapping enzyme (Dcp1), a decapping activator (Pat1/Mrt1) and with mRNA. This indicates that the Lsm proteins may promote decapping by interactions with the mRNA and the decapping machinery. In addition, the Lsm complex that functions in mRNA decay appears to be distinct from the U6-associated Lsm complex, indicating that Lsm proteins form specific complexes that affect different aspects of mRNA metabolism.