Role In Elongation Research Articles

THE RELATIONSHIP OF INTERLEUKIN-6 LEVELS AND THE DEGREE OF MYOPIA IN YOUNG ADULTS IN NORTH SUMATRA UNIVERSITY HOSPITAL

Background.
 Myopia is one of the refractive errors where parallel light from infinity is focused in front of the retina without accommodation. Various cytokines have been implicated in the development of myopia, including interleukin-6 (IL6) and matrix metalloproteinase-2 (MMP-2). IL-6 is one of the pro-inflammatory cytokines that play a role in ocular disease pathology. Chronic subclinical inflammation may play a role in elongation of the eyeball, inflammation promotes the breakdown of the scleral extracellular matrix and results in axial elongation.
 Objective.
 To determine the relationship of interleukin-6 levels and the degree of myopia in young adults in North Sumatra University Hospital.
 Methods.
 This research is an analytic observational study with a case control design, held from February 2022. Primary and secondary data are collected from interviews and direct observation. Bivariate analysis of differences in interleukin-6 levels in myopia and emmetropia uses T-independent test, if the data are normally distributed. If the data are not normally distributed, then the Mann Whitney test is used. Bivariate analysis to analyze differences in interleukin-6 levels based on the degree of myopia uses the Kruskal Wallis test, followed by a post hoc test using the Mann Whitney test and t-independent.
 Results.
 A total sample consists of 30 patients who suffer from myopia and 30 control samples. The mean age in the myopia group was 28.87 years with the youngest age being 25 years and oldest age 34 years. Most of them are moderate myopia with 15 people (50%). There was no significant difference in interleukin-6 levels between myopia and control groups (p = 0.147). There was a difference in the mean level of interleukin-6 based on the degree of myopia (p = 0.005).
 Conclusions.
 There is an association between interleukin-6 levels and the degree of myopia in young adult patients. No differences in interleukin-6 levels were found significant between myopia and control groups.

C-Src–mediated phosphorylation and activation of kinesin KIF1C promotes elongation of invadopodia in cancer cells

Invadopodia on cancer cells play crucial roles in tumor invasion and metastasis by degrading and remodeling the surrounding extracellular matrices and driving cell migration in complex 3D environments. Previous studies have indicated that microtubules (MTs) play a crucial role in elongation of invadopodia, but not their formation, probably by regulating delivery of membrane and secretory proteins within invadopodia. However, the identity of the responsible MT-based molecular motors and their regulation has been elusive. Here, we show that KIF1C, a member of kinesin-3 family, is localized to the tips of invadopodia and is required for their elongation and the invasion of cancer cells. We also found that c-Src phosphorylates tyrosine residues within the stalk domain of KIF1C, thereby enhancing its association with tyrosine phosphatase PTPD1, that in turn activates MT-binding ability of KIF1C, probably by relieving the autoinhibitory interaction between its motor and stalk domains. These findings shed new insights into how c-Src signaling is coupled to the MT-dependent dynamic nature of invadopodia and also advance our understanding of the mechanism of KIF1C activation through release of its autoinhibition.

DOT1L complex regulates transcriptional initiation in human erythroleukemic cells

DOT1L, the only H3K79 methyltransferase in human cells and a homolog of the yeast Dot1, normally forms a complex with AF10, AF17, and ENL or AF9, is dysregulated in most cases of mixed-lineage leukemia (MLLr), and has been believed to regulate transcriptional elongation on the basis of its colocalization with RNA polymerase II (Pol II), the sharing of subunits (AF9 and ENL) between the DOT1L and super elongation complexes, and the distribution of H3K79 methylation on both promoters and transcribed regions of active genes. Here we show that DOT1L depletion in erythroleukemic cells reduces its global occupancy without affecting the traveling ratio or the elongation rate (assessed by 4sUDRB-seq) of Pol II, suggesting that DOT1L does not play a major role in elongation in these cells. In contrast, analyses of transcription initiation factor binding reveal that DOT1L and ENL depletions each result in reduced TATA binding protein (TBP) occupancies on thousands of genes. More importantly, DOT1L and ENL depletions concomitantly reduce TBP and Pol II occupancies on a significant fraction of direct (DOT1L-bound) target genes, indicating a role for the DOT1L complex in transcription initiation. Mechanistically, proteomic and biochemical studies suggest that the DOT1L complex may regulate transcriptional initiation by facilitating the recruitment or stabilization of transcription factor IID, likely in a monoubiquitinated H2B (H2Bub1)-enhanced manner. Additional studies show that DOT1L enhances H2Bub1 levels by limiting recruitment of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. These results advance our understanding of roles of the DOT1L complex in transcriptional regulation and have important implications for MLLr leukemias.

Molecular Insights into FaEG1, a Strawberry Endoglucanase Enzyme Expressed during Strawberry Fruit Ripening.

The endo-β-1,4-glucanases (EGs) that belong to the glycosyl hydrolase family 9 (GH9) have roles in cell wall synthesis, remodeling and degradation. Previous studies have suggested that EGs may play a key role in the ripening of different fruits including strawberries. In this study, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays to determine the transcript accumulation of an endo-β-1,4-glucanase (FaEG1) during fruit development in two different strawberry ‘Camarosa’ and ‘Monterey’ with contrasting softening ratios. Phylogenetic analyses suggest that FaEG1 belongs to the α group of the GH9 family with other proteins previously described with roles in elongation, abscission and ripening. Comparative modeling was used to obtain the FaEG1 structure. The model displays a α-barrel–type structure that is typical of the GH9 enzyme family, and comprises 12 α-helices, 2 310 helices and 6 β-sheets. The catalytic residues were oriented to the solvent in the middle of an open groove. Protein–ligand interactions were explored with cellulose and two xyloglucans as ligands; the results suggest that the FaEG1-cellulose and FaEG1-XXXGXXXG (the most abundant xyloglucan in strawberries) complexes were more stable complexes than XXFGXXFG. The cell wall degradation was observed by scanning electron microscopy (SEM). The data are congruent with the probable role of the FaEG1 protein in the dissembly of the cellulose-hemicellulose fraction during the ripening of strawberry fruit.

In Silico Structural Modeling and Analysis of Elongation Factor-1 Alpha and Elongation Factor-like Protein.

Translation elongation factor-1alpha (EF-1α) or its paralog elongation factor-like proteins (EFL) interact with an aminoacyl-transfer RNA (aa-tRNA) to play its essential role in elongation of peptide-chain during protein synthesis. Species usually have either an EF-1α or EFL protein; however, some species have both EF-1α and EFL (dual-EF-containing species). In the dual-EF-containing species, EF-1α appeared to be highly divergent in the sequence. Homology modeling and surface analysis of EF-1α and EFL were performed to examine the hypothesis that the divergent EF-1α in the dual-EF-containing eukaryotes does not strongly interact with aa-tRNA compared to the canonical EF-1α and EFL. The subsequent molecular dynamics simulations were carried out to confirm the validity of modeled structures and to analyze their stability. It was found that the molecular surfaces of the divergent EF-1α proteins were negatively charged partly, and thus they might not interact with negatively charged aa-tRNA as strongly as the canonical ones. The molecular docking simulations between EF-1α/EFL and aa-tRNA also support the hypothesis.

The Elongator complex regulates hypocotyl growth in darkness and during photomorphogenesis.

The Elongator complex (hereafter Elongator) promotes RNA polymerase II-mediated transcript elongation through epigenetic activities such as histone acetylation. Elongator regulates growth, development, immune response and sensitivity to drought and abscisic acid. We demonstrate that elo mutants exhibit defective hypocotyl elongation but have a normal apical hook in darkness and are hyposensitive to light during photomorphogenesis. These elo phenotypes are supported by transcriptome changes, including downregulation of circadian clock components, positive regulators of skoto- or photomorphogenesis, hormonal pathways and cell wall biogenesis-related factors. The downregulated genes LHY, HFR1 and HYH are selectively targeted by Elongator for histone H3K14 acetylation in darkness. The role of Elongator in early seedling development in darkness and light is supported by hypocotyl phenotypes of mutants defective in components of the gene network regulated by Elongator, and by double mutants between elo and mutants in light or darkness signaling components. A model is proposed in which Elongator represses the plant immune response and promotes hypocotyl elongation and photomorphogenesis via transcriptional control of positive photomorphogenesis regulators and a growth-regulatory network that converges on genes involved in cell wall biogenesis and hormone signaling.This article has an associated First Person interview with the first author of the paper.

The histone variant H2A.Z promotes efficient cotranscriptional splicing in S. cerevisiae.

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from premessenger RNA (pre-mRNA). Recent studies show the processes of RNA synthesis and RNA processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In Saccharomyces cerevisiae, H2A.Z is deposited into chromatin by the SWR-C complex, is found near the 5' ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP (small nuclear RNA [snRNA] plus associated proteins), as H2A.Z shows extensive genetic interactions with U2 snRNP-associated proteins, and RNA sequencing (RNA-seq) reveals that introns with nonconsensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z's role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled, and unspliced RNAs are released. Together, these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

Abstract B21: Exostosin 1 regulates cancer cell stemness in breast cancer cells

Abstract Cancer stem cells (CSCs), a group of cancer cells, are associated with resistance to radiation and chemotherapy and are implicated in recurrent of cancer. Exostosin 1 (EXT1) gene is widely reported as tumor suppressor and its indispensable role in elongation of heparan sulfate (HS) can speculate probable role as tumor promotor. In recent years, a number of tumors are reported to over express EXT1. Here, we investigated the role of EXT1 in the maintenance of cancer cell stemness. MCF7/ADR cells developed by exposing MCF7, breast cancer cells, to doxorubicin for several months in culture, showed high resistance to doxorubicin compared to MCF7, parental cell line. Doxorubicin resistant MCF7/ADR cells have enhanced P-glycoprotein (P-gp) expression. In FACs analysis, MCF7/ADR cells were accounted for high number of CSC population; they possessed large number of ALDH+ and CD44+/CD24- population compared to MCF7. CSC marker, CD44, was found overexpressed in MCF7/ADR compared to MCF7. Besides that, in mammosphere culture, MCF7/ADR cells formed large number of mammospheres in contrast to MCF7. In microarray data analysis, several genes were found upregulated in MCF7/ADR cells and EXT1 was selected as a candidate gene. Overexpressed EXT1 in MCF7/ADR cells was confirmed by real time PCR. The critical role of EXT1 in maintaining cancer cell stemness was confirmed by siRNA mediated knockdown of EXT1. Knockdown of EXT1 repressed CSC markers, reduced population of ALDH+ and CD44+/CD24-, and suppressed expression of CD44. As a molecular mechanism, we detected suppressed expression of heparan sulfate, syndecan1(SDC1) with siEXT1. With these results, we can suggest that EXT1 level might be a determining factor for doxorubicin sensitive breast cancer cells and enhanced expression of EXT1 can take part in development of CSC phenotype on exposure to anthracycline based therapy through upregulation of SDC1. Citation Format: Sarala Manandhar, Chang-gu Kim, Su Young Oh, Sun-Hee Lee, Jiyoon Seok, Yuk-Dong Jung, Hye-Eun Lee, Young-Sun Choi, You Mie Lee*. Exostosin 1 regulates cancer cell stemness in breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B21.

Shewanella oneidensis FabB: A β-ketoacyl-ACP Synthase That Works with C16:1-ACP.

It is established that Escherichia coli β-ketoacyl-ACP synthase (KAS) I (encoded by EcfabB) is the primary, if not exclusive, factor for elongation of the cis-3-decenoyl-ACP (C10:1-ACP) but not effective with C16:1- or longer-chain-ACPs. To test the extent to which these features apply to KAS I proteins in other species, in this study, we examined the physiological role of FabB in Shewanella oneidensis, an excellent model for researching type II fatty acid synthetic (FAS) system and its regulation. We showed that the loss of either FabA (the enzyme that introduces double bond) or FabB, in the absence of DesA which desaturizes C16 and C18 to generate respective C16:1 and C18:1, leads to a UFA auxotroph. However, fatty acid profiles of membrane phospholipid of the fabA and fabB mutants are significantly different, suggesting that FabB participates in steps beyond elongation of C10:1-ACP. Further analyses demonstrated that S. oneidensis FabB differs from EcFabB in that (i) it is not the only enzyme capable of catalyzing elongation of the cis-3-decenoyl-ACP produced by FabA, (ii) it plays a critical role in elongation of C16:1- and longer-chain-ACPs, and (iii) its overproduction is detrimental.

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5'-to-3' "torpedo" exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.

Role of Elongator in Central Nervous System Remyelination.

Role of Elongator in Central Nervous System Remyelination.

Mcm10 deficiency causes defective-replisome-induced mutagenesis and a dependency on error-free postreplicative repair

Mcm10 is a multifunctional replication factor with reported roles in origin activation, polymerase loading, and replication fork progression. The literature supporting these variable roles is controversial, and it has been debated whether Mcm10 has an active role in elongation. Here, we provide evidence that the mcm10–1 allele confers alterations in DNA synthesis that lead to defective-replisome-induced mutagenesis (DRIM). Specifically, we observed that mcm10–1 cells exhibited elevated levels of PCNA ubiquitination and activation of the translesion polymerase, pol-ζ. Whereas translesion synthesis had no measurable impact on viability, mcm10–1 mutants also engaged in error-free postreplicative repair (PRR), and this pathway promoted survival at semi-permissive conditions. Replication gaps in mcm10–1 were likely caused by elongation defects, as dbf4–1 mutants, which are compromised for origin activation did not display any hallmarks of replication stress. Furthermore, we demonstrate that deficiencies in priming, induced by a pol1–1 mutation, also resulted in DRIM, but not in error-free PRR. Similar to mcm10–1 mutants, DRIM did not rescue the replication defect in pol1–1 cells. Thus, it appears that DRIM is not proficient to fill replication gaps in pol1–1 and mcm10–1 mutants. Moreover, the ability to correctly prime nascent DNA may be a crucial prerequisite to initiate error-free PRR.

The roles of the GA receptors GID1a, GID1b, and GID1c in sly1-independent GA signaling

Gibberellin (GA) hormone signaling occurs through proteolytic and non-proteolytic mechanisms. GA binding to the GA receptor GID1 (GA-INSENSITIVE DWARF1) enables GID1 to bind negative regulators of GA responses called DELLA proteins. In proteolytic GA signaling, the SLEEPY1 (SLY1) F-box protein targets DELLA proteins in the GID1-GA-DELLA complex for destruction through the ubiquitin-proteasome pathway. Non-proteolytic GA signaling in sly1 mutants where GA cannot target DELLA proteins for destruction, requires GA and GID1 gene function. Based on comparison of gid1 multiple mutants to sly1 gid1 mutants, GID1a is the primary GA receptor stimulating stem elongation in proteolytic and non-proteolytic signaling, and stimulating fertility in proteolytic GA signaling. GID1b plays the primary role in fertility, and a secondary role in elongation during non-proteolytic GA signaling. The stronger role of GID1b in non-proteolytic GA signaling may result from the fact that GID1b has higher affinity for DELLA protein than GID1a and GID1c.

Grasping with the eyes: The role of elongation in visual recognition of manipulable objects

Processing within the dorsal visual stream subserves object-directed action, whereas visual object recognition is mediated by the ventral visual stream. Recent findings suggest that the computations performed by the dorsal stream can nevertheless influence object recognition. Little is known, however, about the type of dorsal stream information that is available to assist in object recognition. Here, we present a series of experiments that explored different psychophysical manipulations known to bias the processing of a stimulus toward the dorsal visual stream in order to isolate its contribution to object recognition. We show that elongated-shaped stimuli, regardless of their semantic category and familiarity, when processed by the dorsal stream, elicit visuomotor grasp-related information that affects how we categorize manipulable objects. Elongated stimuli may reduce ambiguity during grasp preparation by providing a coarse cue to hand shaping and orientation that is sufficient to support action planning. We propose that this dorsal-stream-based analysis of elongation along a principal axis is the basis for how the dorsal visual object processing stream can affect categorization of manipulable objects.

Estrogen represses gene expression through reconfiguring chromatin structures

Estrogen regulates over a thousand genes, with an equal number of them being induced or repressed. The distinct mechanisms underlying these dual transcriptional effects remain largely unknown. We derived comprehensive views of the transcription machineries assembled at estrogen-responsive genes through integrating multiple types of genomic data. In the absence of estrogen, the majority of genes formed higher-order chromatin structures, including DNA loops tethered to protein complexes involving RNA polymerase II (Pol II), estrogen receptor alpha (ERα) and ERα-pioneer factors. Genes to be ‘repressed’ by estrogen showed active transcription at promoters and throughout the gene bodies; genes to be ‘induced’ exhibited active transcription initiation at promoters, but with transcription paused in gene bodies. In the presence of estrogen, the majority of estrogen-induced genes retained the original higher-order chromatin structures, whereas most estrogen-repressed genes underwent a chromatin reconfiguration. For estrogen-induced genes, estrogen enhances transcription elongation, potentially through recruitment of co-activators or release of co-repressors with unique roles in elongation. For estrogen-repressed genes, estrogen treatment leads to chromatin structure reconfiguration, thereby disrupting the originally transcription-efficient chromatin structures. Our in silico studies have shown that estrogen regulates gene expression, at least in part, through modifying previously assembled higher-order complexes, rather than by facilitating de novo assembly of machineries.

The Arabidopsis Elongator Complex Subunit2 Epigenetically Regulates Plant Immune Responses

The Arabidopsis thaliana Elongator complex subunit2 (ELP2) genetically interacts with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key transcription coactivator of plant immunity, and regulates the induction kinetics of defense genes. However, the mechanistic relationship between ELP2 and NPR1 and how ELP2 regulates the kinetics of defense gene induction are unclear. Here, we demonstrate that ELP2 is an epigenetic regulator required for pathogen-induced rapid transcriptome reprogramming. We show that ELP2 functions in a transcriptional feed-forward loop regulating both NPR1 and its target genes. An elp2 mutation increases the total methylcytosine number, reduces the average methylation levels of methylcytosines, and alters (increases or decreases) methylation levels of specific methylcytosines. Interestingly, infection of plants with the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000/avrRpt2 induces biphasic changes in DNA methylation levels of NPR1 and PHYTOALEXIN DEFICIENT4 (PAD4), which encodes another key regulator of plant immunity. These dynamic changes are blocked by the elp2 mutation, which is correlated with delayed induction of NPR1 and PAD4. The elp2 mutation also reduces basal histone acetylation levels in the coding regions of several defense genes. Together, our data demonstrate a new role for Elongator in somatic DNA demethylation/methylation and suggest a function for Elongator-mediated chromatin regulation in pathogen-induced transcriptome reprogramming.

The SNP in the promoter region of the bovine ELOVL5 gene influences economic traits including subcutaneous fat thickness

Genetic analyses have contributed to improvements of economically important traits derived from adipose tissue such as fatty acid composition in beef. Elongation of very long chain fatty acids (ELOVL) genes encode for the enzymes that play an important role in elongation of long-chain fatty acids. In this study, we aimed to discover genetic polymorphisms of ELOVL gene family in cattle populations to develop genetic markers. As a result, five synonymous mutations were detected in the coding regions of the ELOVL1, ELOVL2, ELOVL3 and ELOVL5 genes. In addition, six mutations were identified in promoter region of the ELOVL5. Two of five mutations in the promoter region of ELOVL5 were expected to alter the ELOVL5 expression and influence the economic traits, because of the high synteny of the region which was essential for activation of Elovl5 in mouse. Therefore, we performed association analysis between the genotypes and traits and our result revealed that T allele of g.-110T>C in ELOVL5 gene promoter indicated significantly thinner subcutaneous fat thickness (TT, 2.39 cm; CT, 2.35 cm) than that of C allele (CC, 2.68 cm) in a Japanese Black population. Our results suggest that the g.-110T>C is a useful genetic marker for the breeding in beef cattle.

Role of Elongation and Secondary Pathways in S6 Amyloid Fibril Growth

The concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.

Changes of large molecular weight hyaluronan and versican in the mouse pubic symphysis through pregnancy.

During pregnancy, the mouse pubic symphysis undergoes expansion and remodeling resulting in formation of a flexible and elastic interpubic ligament allowing passage of a term fetus. In the current study, we sought to identify and characterize components of the extracellular matrix that likely play an important role in elongation and flexibility of the interpubic ligament during parturition. Mouse pubic symphyses and interpubic ligaments collected at time points during pregnancy and postpartum were utilized to evaluate collagen type, collagen content, processing and solubility, matricellular protein, and proteoglycan expression and quantitative assessment of all glycosaminoglycans. These studies revealed increased gene expression for hyaluronan synthase 1, hyaluronan synthase 2, and versican on Gestation Day 18 as well as a decline in protein expression for the versican-degrading protease a disintegrin-like and metalloprotease with thrombospondin type 1 (ADAMTS1) motif. These findings suggest that the primary mediators of increased elongation and flexibility of the interpubic ligament at term result from increased synthesis and reduced metabolism of viscoelasticity-promoting molecules such as high molecular weight hyaluronan and versican.

Eukaryotic translation initiation factor (eIF) 5A stimulates protein synthesis in Saccharomyces cerevisiae

Within the field of eukaryotic protein synthesis, one factor remained putative for decades: eukaryotic translation initiation factor (eIF) 5A. Because eIF5A is an essential protein required for cell proliferation, and one easily targeted by inhibitors, identifying its role in the cell remains important and urgent. Recent reports support early findings that eIF5A stimulates protein synthesis and newly assign the factor a role in elongation rather than initiation. Here we show that eIF5A directly stimulates protein synthesis on native mRNAs, that rapid depletion of eIF5A in vivo immediately leads to a 2-fold inhibition of protein synthesis, and that both the immediate and lasting effects of eIF5A depletion are a reduction in polysome size concomitant with eIF5A depletion. Addition of purified eIF5A to a depleted lysate results in a roughly 2-fold stimulation of protein synthesis in vitro, a result consistent with both older methionyl-puromycin synthesis data and more recently published findings. We find that although eIF5A is not required for protein synthesis, it stimulates the process by about 2- to 3-fold. Our data, along with other published results, reinforce the conclusion that eIF5A stimulates protein synthesis with one important difference: Polysome profiles observed immediately after eIF5A depletion are diagnostic for a role in initiation. This discrepancy is discussed.