A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes

Penzo, Marianna; Carnicelli, Domenica; Montanaro, Lorenzo; Brigotti, Maurizio

doi:10.1038/nprot.2016.072

Cited by 30 publications

(51 citation statements)

References 36 publications

(55 reference statements)

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“…Of note, a similar observation was made with ribosomes carrying an altered ψ content, further strengthening the central role of rRNA modifications in ribosome functioning and translational control [73]. Further exploration of in vitro cell-free assays [72,74] should help decipher which steps of the translation process are controlled by rRNA chemical modifications and identify target mRNAs. Altogether, these studies demonstrate that 2′-O-Me represents a novel molecular support for mRNA translation and contributes to the functional specialization of the ribosome.…”

Section: 2′-o-methylation Contribution To Ribosome Structure and Fmentioning

confidence: 59%

2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

et al. 2018

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Ribosomal RNA (rRNA) undergoes post-transcriptional modification of over 200 nucleotides, predominantly 2′-O-methylation (2′-O-Me). 2′-O-Methylation protects RNA from hydrolysis and modifies RNA strand flexibility but does not contribute to Watson-Crick base pairing. The contribution of 2′-O-Me to the translational capacity of ribosomes has been established. Yet, how 2′-O-Me participates in ribosome biogenesis and ribosome functioning remains unclear. The development of 2′-O-Me quantitative mapping methods has contributed to the demonstration that these modifications are not constitutive but rather provide heterogeneity to the ribosomal population. Moreover, recent advances in ribosome structure analysis and in vitro translation assays have proven, for the first time, that 2′-O-Me contributes to regulating protein synthesis. This review highlights the recent data exploring the impact of 2′-O-Me on ribosome structure and function, and the emerging idea that the rRNA epitranscriptome is involved in translational control.

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Section: 2′-o-methylation Contribution To Ribosome Structure and Fmentioning

confidence: 59%

2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

et al. 2018

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“…To that end, the hybrid in vitro translation technique also called cell-free translation assay [106,107], which is composed of reticulocyte lysates, in which rabbit ribosomes are replaced by ribosomes isolated from cells of interest, provides an optimal model for analyzing intrinsic functional properties of selected ribosome populations. Using purified ribosomes with different rRNA methylation profiles induced by FBL knockdown in cell-free translation assays, it was demonstrated that IRES-dependent translation initiation from IGF1R and some viral RNAs elements, was directly affected by rRNA methylation [98].…”

Section: Impact Of Rrna Modifications On Ribosome-mediated Regulatmentioning

confidence: 99%

Emerging Role of Eukaryote Ribosomes in Translational Control

Venezia

Vincent

Marcel

et al. 2019

IJMS

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Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of “specialized ribosomes”, which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.

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“…From a functional standpoint, the possibility to assay the activity of diversely modified ribosomes might be even more challenging. We have set up a cell free translation system suitable for testing, in a controlled setting, different aspects of the translational activity of ribosomes stringently purified from cultured cells [ 60 ]. Even though this tool is extremely helpful to investigate the functional outcomes of structural alterations in widely represented ribosomal populations within cells, it does not allow evaluating the translational contribution of ribosomal sub-populations that are less abundant.…”

Section: Are Ribosome All Modified At the Same Manner?mentioning

confidence: 99%

Turning Uridines around: Role of rRNA Pseudouridylation in Ribosome Biogenesis and Ribosomal Function

Penzo

Montanaro

2018

Biomolecules

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Ribosomal RNA (rRNA) is extensively edited through base methylation and acetylation, 2′-O-ribose methylation and uridine isomerization. In human rRNA, 95 uridines are predicted to by modified to pseudouridine by ribonucleoprotein complexes sharing four core proteins and differing for a RNA sequence guiding the complex to specific residues to be modified. Most pseudouridylation sites are placed within functionally important ribosomal domains and can influence ribosomal functional features. Information obtained so far only partially explained the degree of regulation and the consequences of pseudouridylation on ribosomal structure and function in different physiological and pathological conditions. This short review focuses on the available evidence in this topic, highlighting open questions in the field and perspectives that the development of emerging techniques is offering.

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A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes

Cited by 30 publications

References 36 publications

2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

2′-O-Methylation of Ribosomal RNA: Towards an Epitranscriptomic Control of Translation?

Emerging Role of Eukaryote Ribosomes in Translational Control

Turning Uridines around: Role of rRNA Pseudouridylation in Ribosome Biogenesis and Ribosomal Function

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