Biology subjects

Hellen, C. U. T.

Publications and source records attributed to Hellen, C. U. T..

9 recordsLinked to original sources

bioRxiv2025

Structural Studies of Nedicistrovirus IRES-Driven, Initiation Factor-independent Translation Shed Light on Key Steps of Eukaryotic Translation Elongation

We utilized the Nedicistrovirus (NediV) intergenic region (IGR) IRES-mediated, initiation factor-independent translation initiation system and determined high-resolution structures of 80S ribosome complexes with the NediV IRES in various functional states, including binary complexes, aminoacyl-tRNA-bound complexes, and complexes with elongation factor eEF2. In binary complexes, the NediV IRES primarily occupies the ribosomal P site, exhibiting conformational flexibility and engaging the ribosome at multiple interaction sites. Upon translocation, the IRES undergoes structural rearrangements, including destabilization of its PKI domain, facilitating the transition to canonical elongation. Crucially, we captured an eEF2-bound complex, along with an eEF1A-bound post-proofreading complex featuring a mismatched tRNA, the latter representing the first instance of a canonical elongation complex visualized in the presence of a natural, hydrolysable nucleotide and without the addition of any trapping agents. These findings provide a comprehensive structural overview of IGR IRES-mediated translation initiation and its transition to elongation, revealing key mechanistic details of viral translation and proofreading.

biochemistry↗

bioRxiv2025

The translation initiation factor DHX29 appears to pull on mRNA in a direction opposite to scanning.

The DExH-box helicase DHX29 plays a critical role in mammalian translation initiation. It is required for the scanning of mRNAs with complex 5UTRs. Despite its importance, the detailed mechanism of DHX29s action has remained debated. Using structural models derived from AlphaFold and cryo-EM structure of DHX29 bound to the ribosomal 43S pre-initiation complex, we provide a revised structural framework that clarifies the interplay between DHX29, the 40S ribosomal subunit, and eIF3. Our findings suggest that the 40S subunit regulates DHX29s NTPase activity through an activation mechanism resembling the G-patch protein regulation of DEAH helicases. Moreover, our model supports a 3 to 5 translocase mechanism, in which DHX29 transiently pulls the mRNA opposite to the scanning direction, destabilizing stable stem-loops trapped in the mRNA channel and allowing scanning to resume. This structural analysis refines our understanding of DHX29s function and provides new hypotheses regarding its role in mRNA unwinding during scanning and start codon selection.

biophysics↗

bioRxiv2025

The mechanism of ribosomal recruitment during translation initiation on Type 2 IRESs

The encephalomyocarditis virus (EMCV) IRES and other Type 2 IRESs comprise domains H-L and specifically interact with eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES function, which also requires the preceding domain I of unknown function. To identify interactions that drive ribosomal recruitment of eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. It revealed that the apical domain I cloverleaf contacts ribosomal proteins uS13 and uS19 via its Id subdomain and that the essential GNRA tetraloop in subdomain Ic interacts directly with the T{psi}C domain of initiator tRNA. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of primary and secondary structures of the apex of domain I among Type 2 IRESs suggests that the reported interactions are a common essential feature of them all.

molecular biology↗

bioRxiv2024

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome- associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2[~]Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48- specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25s action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48-specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1[~]Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.

molecular biology↗

bioRxiv2024

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institutes rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

biophysics↗

bioRxiv2024

Genetic mechanisms underlying the structural elaboration and dissemination of viral internal ribosomal entry sites

Viral internal ribosomal entry sites (IRESs) form several classes that use distinct mechanisms to mediate end-independent initiation of translation. The origin of viral IRESs is a longstanding question. The simplest IRESs comprise tandem pseudoknots and occur in the intergenic region (IGR) of Dicistroviridae genomes (order Picornavirales). Larger IGR IRESs contain additional elements that determine specific properties such as binding to the head of the ribosoma l 40S subunit. Metagenomic analyses reported here identified novel groups of structurally distinct IGR-like IRESs. The smallest of these ([~]120nt long) comprise three pseudoknots and bind directly to the ribosomal P site. Others are up to 260nt long: insertions occurred at specific loci, possibly reflecting non-templated nucleotide insertion during replication. Various groups can be arranged in order, differing by the cumulative addition of single structural elements, suggesting an accretion mechanism for the structural elaboration of IRESs. Identification of chimeric IRESs implicates recombinational exchange of domains as a second mechanism for the diversification of IRES structure. Recombination likely also accounts for the presence of IGR-like IRESs at the 5-end of some dicistrovirus-like genomes (e.g. Hangzhou dicistrovirus 3) and in the RNA genomes of Tombusviridae (order Tolivirales), Marnaviridae (order Picornavirales), and the Ripiresk picorna-like clade (order Picornavirales).

molecular biology↗

bioRxiv2023

In vitro reconstitution of SARS CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on {beta}-globin, EMCV IRES and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3gs RRM domain. The cleavage site was located in the coding region 18 nucleotides downstream from the mRNA entrance indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1s N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3gs RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of Nsp1-NTD and eIF3gs RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

molecular biology↗

bioRxiv2021

Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 [A] that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

molecular biology↗

bioRxiv2020

The Halastavi arva virus intergenic region IRES promotes translation by the simplest possible initiation mechanism

Dicistrovirus intergenic region internal ribosomal entry sites (IGR IRES) do not require initiator tRNA, an AUG codon or initiation factors, and jumpstart translation from the middle of the elongation cycle via formation of IRES/80S complexes resembling the pre-translocation state. eEF2 then translocates the [codon-anticodon]-mimicking pseudoknot I (PKI) from ribosomal A to P sites, bringing the first sense codon into the decoding center. Halastavi arva virus (HalV) contains an IGR that is related to previously described IGR IRESs, but lacks domain 2, which enables these IRESs to bind to individual 40S ribosomal subunits. By employing in vitro reconstitution and cryo-electron microscopy, we now report that the HalV IGR IRES functions by the simplest initiation mechanism that involves binding to 80S ribosomes such that PKI is placed in the P site, so that the A site contains the first codon that is directly accessible for decoding without prior eEF2-mediated translocation of PKI.

molecular biology↗