Translational Regulation in Arabidopsis thaliana: Genetic and Functional Characterization of Eukaryotic Initiation Factor 3

Molecular functions of eukaryotic initiation factor 3 (eIF3) in translation are manifold, encompassing events from initiation complex assembly to translation termination. The contribution of the individual subunits of eIF3 to its multiple activities is quite unclear. It has been hypothesized that several of its 13 subunits contribute to mRNA specific regulation. Prior research had established that the h subunit of eIF3 in Arabidopsis was required for translation of specific mRNAs as well as for organ formation and meristem development. This study aims towards understanding the functions of individual subunits of eIF3 in the context of plant development and to further define the role of eIF3h at the molecular level. This dissertation describes an effort to identify mutations affecting each of the 13 eIF3 subunits. Using a panel of pollen-specific fluorescent marker genes, eIF3 subunits e, h and i1 were demonstrated to be essential for normal male gametophyte development. Furthermore, subunits b and c proved to be essential for embryo development. In contrast, a mutation in eIF3k revealed no phenotypic abnormalities. This work represents a systematic effort to attribute functions to many of the eIF3 subunits in growth and development in a multicellular eukaryote. The h subunit of eIF3 is necessary for the efficient translation of specific mRNAs in Arabidopsis. In particular, eIF3h fosters the translation of those mRNAs that harbor multiple upstream open reading frames (uORFs) in their 5’ leader. The specific molecular activity of eIF3h was investigated by structure-function analysis of the 5\u27 leader of the Arabidopsis AtbZip11 mRNA, which harbors a set of four uORFs that is evolutionarily conserved. By pairing extensive mutagenesis of the AtbZip11 5\u27 leader with gene expression analysis in Arabidopsis seedlings, it was revealed that eIF3h helps the ribosome to retain its reinitiation competence during uORF translation. These data establish a function for the h subunit of eIF3 in a special case of translation initiation, reinitiation. Finally, the molecular events during translation reinitiation were investigated further for a functional cooperation between eIF3h and the large subunit of the ribosome, given that the large ribosomal subunit had been implicated in reinitiation in other biological contexts. Reinitiation profiling using the AtbZip11 leader demonstrated that a protein of the large ribosomal subunit, RPL24B, bolsters reinitiation similar to eIF3h. Taken together, there exists a functional cooperation between the large ribosomal subunit and eIF3 that helps ribosomes to reinitiate after translation of uORFs

Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24

Background Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs). Results Here we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings. Conclusion We conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORF-containing mRNAs and, in turn, to orchestrate plant development. doi:10.1186/1471-2229-10-19

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3\u27 end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3\u27 UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD)

Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression

A premature termination codon (PTC) in the ORF of an mRNA generally leads to production of a truncated polypeptide, accelerated degradation of the mRNA, and depression of overall mRNA expression. Accordingly, nonsense mutations cause some of the most severe forms of inherited disorders. The small-molecule drug ataluren promotes therapeutic nonsense suppression and has been thought to mediate the insertion of near-cognate tRNAs at PTCs. However, direct evidence for this activity has been lacking. Here, we expressed multiple nonsense mutation reporters in human cells and yeast and identified the amino acids inserted when a PTC occupies the ribosomal A site in control, ataluren-treated, and aminoglycoside-treated cells. We find that ataluren\u27s likely target is the ribosome and that it produces full-length protein by promoting insertion of near-cognate tRNAs at the site of the nonsense codon without apparent effects on transcription, mRNA processing, mRNA stability, or protein stability. The resulting readthrough proteins retain function and contain amino acid replacements similar to those derived from endogenous readthrough, namely Gln, Lys, or Tyr at UAA or UAG PTCs and Trp, Arg, or Cys at UGA PTCs. These insertion biases arise primarily from mRNA:tRNA mispairing at codon positions 1 and 3 and reflect, in part, the preferred use of certain nonstandard base pairs, e.g., U-G. Ataluren\u27s retention of similar specificity of near-cognate tRNA insertion as occurs endogenously has important implications for its general use in therapeutic nonsense suppression

Fluorescence-Tagged Transgenic Lines Reveal Genetic Defects in Pollen Growth—Application to the Eif3 Complex

BACKGROUND: Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis. METHODOLOGY/PRINCIPAL FINDINGS: Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation. CONCLUSION/SIGNIFICANCE: eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis

Translational Regulation of Cytoplasmic mRNAs

Translation of the coding potential of a messenger RNA into a protein molecule is a fundamental process in all living cells and consumes a large fraction of metabolites and energy resources in growing cells. Moreover, translation has emerged as an important control point in the regulation of gene expression. At the level of gene regulation, translational control is utilized to support the specific life histories of plants, in particular their responses to the abiotic environment and to metabolites. This review summarizes the diversity of translational control mechanisms in the plant cytoplasm, focusing on specific cases where mechanisms of translational control have evolved to complement or eclipse other levels of gene regulation. We begin by introducing essential features of the translation apparatus. We summarize early evidence for translational control from the pre-Arabidopsis era. Next, we review evidence for translation control in response to stress, to metabolites, and in development. The following section emphasizes RNA sequence elements and biochemical processes that regulate translation. We close with a chapter on the role of signaling pathways that impinge on translation

Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24

Abstract Background Within the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs). Results Here we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings. Conclusion We conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORF-containing mRNAs and, in turn, to orchestrate plant development.</p

translation of mRNAs harboring upstream open reading frames The h subunit of eIF3 promotes reinitiation competence during References http://rnajournal.cshlp.org/content/16/4/748.full.html#ref-list-1 The h subunit of eIF3 promotes reinitiation competence d

ABSTRACT Upstream open reading frames (uORFs) are protein coding elements in the 59 leader of messenger RNAs. uORFs generally inhibit translation of the main ORF because ribosomes that perform translation elongation suffer either permanent or conditional loss of reinitiation competence. After conditional loss, reinitiation competence may be regained by, at the minimum, reacquisition of a fresh methionyl-tRNA. The conserved h subunit of Arabidopsis eukaryotic initiation factor 3 (eIF3) mitigates the inhibitory effects of certain uORFs. Here, we define more precisely how this occurs, by combining gene expression data from mutated 59 leaders of Arabidopsis AtbZip11 (At4g34590) and yeast GCN4 with a computational model of translation initiation in wild-type and eif3h mutant plants. Of the four phylogenetically conserved uORFs in AtbZip11, three are inhibitory to translation, while one is anti-inhibitory. The mutation in eIF3h has no major effect on uORF start codon recognition. Instead, eIF3h supports efficient reinitiation after uORF translation. Modeling suggested that the permanent loss of reinitiation competence during uORF translation occurs at a faster rate in the mutant than in the wild type. Thus, eIF3h ensures that a fraction of uORF-translating ribosomes retain their competence to resume scanning. Experiments using the yeast GCN4 leader provided no evidence that eIF3h fosters tRNA reaquisition. Together, these results attribute a specific molecular function in translation initiation to an individual eIF3 subunit in a multicellular eukaryote

Yeast Upf1 CH domain interacts with Rps26 of the 40S ribosomal subunit

The central nonsense-mediated mRNA decay (NMD) regulator, Upf1, selectively targets nonsense-containing mRNAs for rapid degradation. In yeast, Upf1 preferentially associates with mRNAs that are NMD substrates, but the mechanism of its selective retention on these mRNAs has yet to be elucidated. Previously, we demonstrated that Upf1 associates with 40S ribosomal subunits. Here, we define more precisely the nature of this association using conventional and affinity-based purification of ribosomal subunits, and a two-hybrid screen to identify Upf1-interacting ribosomal proteins. Upf1 coimmunoprecipitates specifically with epitope-tagged 40S ribosomal subunits, and Upf1 association with high-salt washed or puromycin-released 40S subunits was found to occur without simultaneous eRF1, eRF3, Upf2, or Upf3 association. Two-hybrid analyses and in vitro binding assays identified a specific interaction between Upf1 and Rps26. Using mutations in domains of UPF1 known to be crucial for its function, we found that Upf1:40S association is modulated by ATP, and Upf1:Rps26 interaction is dependent on the N-terminal Upf1 CH domain. The specific association of Upf1 with the 40S subunit is consistent with the notion that this RNA helicase not only triggers rapid decay of nonsense-containing mRNAs, but may also have an important role in dissociation of the premature termination complex