Biology subjects

Gordiyenko, Y.

Publications and source records attributed to Gordiyenko, Y..

8 recordsLinked to original sources

bioRxiv2025

Mechanism of translation initiation on endogenous eukaryotic circular RNAs

Eukaryotic circular RNAs (circRNAs) perform a wide variety of functions. A subset of circRNAs has been demonstrated to undergo translation in vivo. However, few insights exist on the underlying translation mechanisms. Here, we elucidate the basis of translation initiation in two naturally occurring circular RNAs, circMbl and circSfl. We show that in vitro prepared versions of both circRNAs are translated in eukaryotic cell lysates and cells. Initiation depends on the untranslated region (UTR) and can be reconstituted using only the 43S pre-initiation complex, eukaryotic initiation factors 4G, 4A, 4B, and, for circSfl, the RNA helicase DHX29. Functional assays and structural analysis of the initiation complexes suggest that scanning until recognition of the start codon follows initial ribosome landing occurring on AU-rich, accessible UTR patches upstream of the initiation sites. Together, these results provide key insights into how eukaryotic ribosomes engage with and initiate translation on circular endogenous transcripts.

molecular biology↗

bioRxiv2025

Structure of a transcribing Pol II-DSIF-SPT6-U1 snRNP complex

RNA polymerase II (Pol II) facilitates co-transcriptional splicing by recruiting the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to the nascent transcripts. Here, we report the cryo-electron microscopy structure of a transcribing Pol II-U1 snRNP complex with elongation factors DSIF and SPT6. Furthermore, our biochemical analysis revealed that the phosphorylated Pol II carboxyl-terminal domain and SPT6 interact directly with U1 snRNP proteins, facilitating its recruitment to the elongation complex. This multivalent interaction allows efficient spliceosome assembly and ensures transcription processivity.

molecular biology↗

bioRxiv2025

Structural basis of RECQL5-induced RNA polymerase II transcription braking and subsequent reactivation

During productive transcription elongation, the speed of RNA polymerase II (Pol II) is highly dynamic within individual genes and varies between different genes1,2. Unregulated rapid transcription elongation can lead to detrimental consequences such as transcription-replication collisions, altered alternative splicing patterns, and genome instability1-7. Therefore, elongating Pol II requires mechanisms to slow its progression, yet the molecular basis of transcription braking remains unclear. RECQL5 is a DNA helicase that functions as a general elongation factor by slowing down Pol II8-11. Here we report cryo-electron microscopy (cryo-EM) structures of human RECQL5 bound to multiple transcription elongation complexes. Combined with biochemical analysis, we identify an -helix of RECQL5 responsible for Pol II binding and slowdown of transcription elongation. We further reveal that the transcription-coupled DNA repair (TCR) complex allows Pol II to overcome RECQL5-induced transcription braking through concerted actions of its translocase activity and competition with RECQL5 for engaging Pol II. Additionally, RECQL5 inhibits TCR-mediated Pol II ubiquitination to prevent activation of the DNA repair pathway. Our results suggest a model in which RECQL5 and the TCR complex coordinately regulate the transcription elongation rate to ensure transcription efficiency while maintaining genome stability. This work provides a framework for future studies on the regulatory role of elongation speed in gene expression.

molecular biology↗

bioRxiv2024

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in 40S small ribosomal subunit

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

biochemistry↗

bioRxiv2022

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

SummaryInitiation of mRNA translation is a key regulatory step in gene expression in all eukaryotes. Canonical initiation of translation in eukaryotes involves recruitment of the 43S preinitiation complex to the 5' end of mRNA by the cap-binding complex eIF4F to form the 48S initiation complex (48S), followed by scanning along the mRNA until the start codon is selected.1-8 We have previously shown that eIF4F binds near the mRNA channel exit site of the 43S, leaving an open question about how mRNA secondary structure is removed as it enters the mRNA binding channel on the other side of the 40S subunit.4 Here we describe a human 48S positioned at the start codon that shows that in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound to the mRNA entry site. The entry channel bound eIF4A is positioned through interactions with eIF3 and the 40S subunit to enable its ATP-dependent helicase activity to directly unwind secondary structure located downstream of the scanning 48S complex. The structure also reveals universally conserved interactions between eIF4F and the 48S, likely explaining how this complex can promote mRNA recruitment in all eukaryotes. mRNA translation has emerged as an important tool for developing innovative therapies, yet several fundamental aspects of its regulation remain unknown. This work sheds light on the critical regulatory roles of eIF4A and eIF4F during the recruitment and scanning of the 5' UTR of mRNA.

molecular biology↗

bioRxiv2018

Structural basis for the inhibition of translation through eIF2α phosphorylation

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting its interaction with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.15 [A]. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2-D1, which contains the phosphorylated Ser51. eIF2-D1 is mainly inserted between the N-terminal helix bundle domains of {delta} and subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2B and indirectly by inducing contacts of eIF2 helix 58-63 with eIF2B{delta} leading to a competition with Met-tRNAi.

biophysics↗

bioRxiv2018

The beta propellers of eIF3b and eIF3i relocate together to the ribosomal intersubunit interface during translation initiation

During eukaryotic translational initiation, the 48S ribosomal pre-initiation complex (PIC) scans the 5 untranslated region of mRNA until it encounters a start codon. We present a single particle electron cryomicroscopy (cryo-EM) reconstruction of a yeast 48S PIC in an open scanning-competent state in which eIF3b is observed bound on the 40S subunit interface. eIF3b is re-located with eIF3i from their solvent-interface locations observed in other PIC structures; however, eIF3i is not in contact with the 40S. Re-processing of micrographs of our previous 48S PIC in a closed state using currently available tools reveal a similar re-location of eIF3b and eIF3i from the solvent to subunit interface. Genetic analysis indicates that high fidelity initiation in vivo depends strongly on eIF3b interactions at the subunit interface that either promote the closed conformation of the PIC on start codon selection or facilitate subsequent relocation back to the solvent side of the 40S subunit.

molecular biology↗

bioRxiv2018

Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition

In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNAi in a \"PIN\" state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 [A], featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.

biophysics↗