Structural insights into the mammalian late-stage initiation complexes

SUMMARYIn higher eukaryotes, the mRNA sequence in direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S IC) in the presence of two different native mRNA sequences, β-globin and histone 4 (H4) at overall resolution of 3Å and 3.5Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIF): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we were able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of the ribosome/eIFs –mRNA and –tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.

Download Full-text

Structural insights and evaluation of the potential impact of missense variants on the interactions of SLIT2 with ROBO1/4 in cancer progression

Scientific Reports ◽

10.1038/s41598-020-78882-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Debmalya Sengupta ◽

Gairika Bhattacharya ◽

Sayak Ganguli ◽

Mainak Sengupta

Keyword(s):

Lung Cancer ◽

Cancer Progression ◽

Structural Parameters ◽

Homology Modelling ◽

Missense Variants ◽

Potential Impact ◽

The Impact ◽

Structural Insights ◽

Rigid Docking

AbstractThe cognate interaction of ROBO1/4 with its ligand SLIT2 is known to be involved in lung cancer progression. However, the precise role of genetic variants, disrupting the molecular interactions is less understood. All cancer-associated missense variants of ROBO1/4 and SLIT2 from COSMIC were screened for their pathogenicity. Homology modelling was done in Modeller 9.17, followed by molecular simulation in GROMACS. Rigid docking was performed for the cognate partners in PatchDock with refinement in HADDOCK server. Post-docking alterations in conformational, stoichiometric, as well as structural parameters, were assessed. The disruptive variants were ranked using a weighted scoring scheme. In silico prioritisation of 825 variants revealed 379 to be potentially pathogenic out of which, about 12% of the variants, i.e. ROBO1 (14), ROBO4 (8), and SLIT2 (23) altered the cognate docking. Six variants of ROBO1 and 5 variants of ROBO4 were identified as "high disruptors" of interactions with SLIT2 wild type. Likewise, 17 and 13 variants of SLIT2 were found to be "high disruptors" of its interaction with ROBO1 and ROBO4, respectively. Our study is the first report on the impact of cancer-associated missense variants on ROBO1/4 and SLIT2 interactions that might be the drivers of lung cancer progression.

Download Full-text

The role of IRES trans-acting factors in regulating translation initiation

Biochemical Society Transactions ◽

10.1042/bst0381581 ◽

2010 ◽

Vol 38 (6) ◽

pp. 1581-1586 ◽

Cited By ~ 73

Author(s):

Helen A. King ◽

Laura C. Cobbold ◽

Anne E. Willis

Keyword(s):

Translation Initiation ◽

Alternative Form ◽

Structural Element ◽

Entry Site ◽

Internal Ribosome Entry ◽

Cis Acting ◽

Initiation Factors ◽

Eukaryotic Initiation Factors ◽

Cellular Pathways

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.

Download Full-text

Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release

Nature Communications ◽

10.1038/s41467-021-27257-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wanqiu Li ◽

Linlin Wang ◽

Bradley M. Wierbowski ◽

Mo Lu ◽

Feitong Dong ◽

...

Keyword(s):

Small Molecule ◽

Extracellular Domain ◽

Proteolytic Cleavage ◽

Proteolytic Activation ◽

Atomic Structures ◽

Cryo Electron Microscopy ◽

Extracellular Release ◽

Before And After ◽

Structural Insights

AbstractThe membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1–Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.

Download Full-text

Dendritic Targeting and Regulatory RNA Control of Local Neuronal Translation

The Oxford Handbook of Neuronal Protein Synthesis ◽

10.1093/oxfordhb/9780190686307.013.8 ◽

2018 ◽

Cited By ~ 1

Author(s):

Taesun Eom ◽

Ilham A. Muslimov ◽

Anna Iacoangeli ◽

Henri Tiedge

Keyword(s):

Translational Control ◽

Translational Regulation ◽

Cognitive Disorders ◽

Initiation Factors ◽

Brain Functions ◽

Eukaryotic Initiation Factors ◽

Rna Targeting ◽

Dendritic Rna ◽

Future Work

This chapter reviews current developments in the area of translational control in neurons. It focuses on the activity-dependent translational modulation by neuronal regulatory RNAs, including underlying interactions with eukaryotic initiation factors (eIFs), and on the role of such modulation in locally controlled protein synthesis in synapto-dendritic domains. It highlights the role of dendritic RNA targeting as a key prerequisite of local translation at the synapse and discusses the significance of these mechanisms in the expression of higher brain functions, including learning, memory, and cognition. The chapter concludes with discussion of anticipated future work to continue to elucidate these mechanisms and provide advances in the area of translational regulation in neurons and our understanding of how translational dysregulation contributes to neurological and cognitive disorders.

Download Full-text

The Jigsaw Puzzle of mRNA Translation Initiation in Eukaryotes: A Decade of Structures Unraveling the Mechanics of the Process

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070816-034034 ◽

2018 ◽

Vol 47 (1) ◽

pp. 125-151 ◽

Cited By ~ 18

Author(s):

Yaser Hashem ◽

Joachim Frank

Keyword(s):

Structural Biology ◽

Translation Initiation ◽

Mrna Translation ◽

Start Codon ◽

Jigsaw Puzzle ◽

Reading Frame ◽

Eukaryotic Translation ◽

Initiation Factors ◽

Eukaryotic Initiation Factors ◽

Initiation Process

Translation initiation in eukaryotes is a highly regulated and rate-limiting process. It results in the assembly and disassembly of numerous transient and intermediate complexes involving over a dozen eukaryotic initiation factors (eIFs). This process culminates in the accommodation of a start codon marking the beginning of an open reading frame at the appropriate ribosomal site. Although this process has been extensively studied by hundreds of groups for nearly half a century, it has been only recently, especially during the last decade, that we have gained deeper insight into the mechanics of the eukaryotic translation initiation process. This advance in knowledge is due in part to the contributions of structural biology, which have shed light on the molecular mechanics underlying the different functions of various eukaryotic initiation factors. In this review, we focus exclusively on the contribution of structural biology to the understanding of the eukaryotic initiation process, a long-standing jigsaw puzzle that is just starting to yield the bigger picture.

Download Full-text

Intergroup Threat and Outgroup Attitudes

Social Psychology ◽

10.1027/1864-9335/a000127 ◽

2013 ◽

Vol 44 (5) ◽

pp. 311-319 ◽

Cited By ~ 12

Author(s):

Marco Brambilla ◽

David A. Butz

Keyword(s):

Gay Men ◽

Social Group ◽

Welfare Recipients ◽

Intergroup Attitudes ◽

Social Policies ◽

Intergroup Threat ◽

Economic Threat ◽

The Impact ◽

General Climate

Two studies examined the impact of macrolevel symbolic threat on intergroup attitudes. In Study 1 (N = 71), participants exposed to a macrosymbolic threat (vs. nonsymbolic threat and neutral topic) reported less support toward social policies concerning gay men, an outgroup whose stereotypes implies a threat to values, but not toward welfare recipients, a social group whose stereotypes do not imply a threat to values. Study 2 (N = 78) showed that, whereas macrolevel symbolic threat led to less favorable attitudes toward gay men, macroeconomic threat led to less favorable attitudes toward Asians, an outgroup whose stereotypes imply an economic threat. These findings are discussed in terms of their implications for understanding the role of a general climate of threat in shaping intergroup attitudes.

Download Full-text