bims-tricox 2022-11-06 papers

Issue of 2022‒11‒06
five papers selected by
Yash Verma
University of Delhi South Campus

RNA. 2022 Nov 02. pii: rna.079149.122. [Epub ahead of print]

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing.

Mason A McCool, Amber F Buhagiar, Carson J Bryant, Lisa M Ogawa, Laura Abriola, Yulia V Surovtseva, Susan J Baserga.

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for 60S ribosomal subunit biogenesis. In addition, the PeBoW (BOP1-PES1-WDR12) complex has been well-defined as required for pre-28S rRNA processing and cell proliferation. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in human cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We demonstrate that RSL24D1 and WDR12 co-immunoprecipitate with the RNA polymerase I subunit, RPA194, and regulate its steady state levels. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large ribosomal subunit biogenesis factors in pre-rRNA transcription control.

Keywords: 60S large ribosomal subunit; nucleolus; pre-rRNA processing; rRNA transcription; ribosome biogenesis

DOI: https://doi.org/10.1261/rna.079149.122

Biochim Biophys Acta Gene Regul Mech. 2022 Oct 31. pii: S1874-9399(22)00105-5. [Epub ahead of print] 194890

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein.

Kamil Filipek, Kamil Deryło, Barbara Michalec-Wawiórka, Monika Zaciura, Alan González, Dawid Krokowski, Przemysław Latoch, Agata L Starosta, Jakub Czapiński, Adolfo Rivero-Müller, Leszek Wawiórka, Marek Tchórzewski.

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10β. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10β protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10β is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10β undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10β, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

Keywords: Alternative splicing; ER stress; Extra-ribosomal functions; GTP-associated center; Ribosomal P-stalk; Ribosomal protein isoforms; Ribosome; Specialized ribosomes

DOI: https://doi.org/10.1016/j.bbagrm.2022.194890

Nucleic Acids Res. 2022 Nov 02. pii: gkac961. [Epub ahead of print]

The nucleoplasmic phase of pre-40S formation prior to nuclear export.

Jingdong Cheng, Benjamin Lau, Matthias Thoms, Michael Ameismeier, Otto Berninghausen, Ed Hurt, Roland Beckmann.

Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23-TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.

DOI: https://doi.org/10.1093/nar/gkac961

Nat Chem. 2022 Oct 31.

Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.

Egor A Syroegin, Elena V Aleksandrova, Yury S Polikanov.

During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform β-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.

DOI: https://doi.org/10.1038/s41557-022-01073-1

PLoS Genet. 2022 Oct 31. 18(10): e1010460

Translational buffering by ribosome stalling in upstream open reading frames.

Ty A Bottorff, Heungwon Park, Adam P Geballe, Arvind Rasi Subramaniam.

Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames through several mechanisms: siphoning away scanning ribosomes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5' UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF protein expression when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF protein expression via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.

DOI: https://doi.org/10.1371/journal.pgen.1010460