scholarly journals RiboTRIBE: Monitoring Translation with ADAR-meditated RNA Editing

2021 ◽  
Author(s):  
Weijin Xu ◽  
Michael Rosbash
Keyword(s):  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Catalytic Domain ◽  
Small Subunit ◽  
Ribosome Profiling ◽  
S2 Cells ◽  
Mrna Editing ◽  
Rna Translation ◽  
Biochemical Assays ◽  
Drosophila S2 Cells

RNA translation is tightly regulated to ensure proper protein expression in cells and tissues. Translation is often assayed with biochemical assays such as ribosome profiling and TRAP, which are effective in many contexts. These assays are however not ideal with limiting amounts of biological material when it can be difficult or even impossible to make an extract with sufficient signal or sufficient signal:noise. Because of our interest in translational regulation within the few Drosophila adult circadian neurons, we fused the ADAR catalytic domain (ADARcd) to several small subunit ribosomal proteins and assayed mRNA editing in Drosophila S2 cells. The strategy is named RiboTRIBE and is analogous to a recently published APOBEC-based method. The list of RiboTRIBE-edited transcripts overlaps well with ribosome profiling targets, especially with more highly ranked targets. There is also an enriched number of editing sites in ribosome-associated mRNA comparing to total mRNA, indicating that editing occurs preferentially on polyribosome-associated transcripts. The use of cycloheximide to freeze translating ribosomes causes a substantial increase in the number of RiboTRIBE targets, which is decreased by pretreating cells with the chain terminating drug puromycin. The data taken together indicate that RiboTRIBE successfully identifies transcripts undergoing active translation.

scholarly journals Decoding regulatory specificity of human ribosomal proteins on global gene expression

2021 ◽  
Author(s):  
Yizhao Luan ◽  
Nan Tang ◽  
Jiaqi Yang ◽  
Shuting Liu ◽  
Chichi Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Large Subunit ◽  
Global Gene Expression ◽  
Small Subunit ◽  
Sequencing Analysis ◽  
Retina Development ◽  
P53 Signaling

Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with few regulatory functions. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is unclear. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We assessed deficiency of 75 RP, including 44 from the large subunit (60S) and 31 from the small subunit (40S), on gene translation and transcription via ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S or the 40S RPs had distinguished effects on the two subunits. The gene ontology analysis revealed that RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by affecting P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis but RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity and provided an important resource which offered novel insights into ribosome regulation in human diseases and cancer.

scholarly journals Selective Translation of Low Abundance and Upregulated Transcripts in Halobacterium salinarum

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Adrián López García de Lomana ◽  
Ulrike Kusebauch ◽  
Arjun V. Raman ◽  
Min Pan ◽  
Serdar Turkarslan ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Physiological State ◽  
Protein Composition ◽  
Ribosome Profiling ◽  
Halobacterium Salinarum ◽  
Active Growth ◽  
Content Type ◽  
Growth State ◽  
Selective Translation

ABSTRACT When organisms encounter an unfavorable environment, they transition to a physiologically distinct, quiescent state wherein abundant transcripts from the previous active growth state continue to persist, albeit their active transcription is downregulated. In order to generate proteins for the new quiescent physiological state, we hypothesized that the translation machinery must selectively translate upregulated transcripts in an intracellular milieu crowded with considerably higher abundance transcripts from the previous active growth state. Here, we have analyzed genome-wide changes in the transcriptome (RNA sequencing [RNA-seq]), changes in translational regulation and efficiency by ribosome profiling across all transcripts (ribosome profiling [Ribo-seq]), and protein level changes in assembled ribosomal proteins (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) to investigate the interplay of transcriptional and translational regulation in Halobacterium salinarum as it transitions from active growth to quiescence. We have discovered that interplay of regulatory processes at different levels of information processing generates condition-specific ribosomal complexes to translate preferentially pools of low abundance and upregulated transcripts. Through analysis of the gene regulatory network architecture of H. salinarum, Escherichia coli, and Saccharomyces cerevisiae, we demonstrate that this conditional, modular organization of regulatory programs governing translational systems is a generalized feature across all domains of life. IMPORTANCE Our findings demonstrate conclusively that low abundance and upregulated transcripts are preferentially translated, potentially by environment-specific translation systems with distinct ribosomal protein composition. We show that a complex interplay of transcriptional and posttranscriptional regulation underlies the conditional and modular regulatory programs that generate ribosomes of distinct protein composition. The modular regulation of ribosomal proteins with other transcription, translation, and metabolic genes is generalizable to bacterial and eukaryotic microbes. These findings are relevant to how microorganisms adapt to unfavorable environments when they transition from active growth to quiescence by generating proteins from upregulated transcripts that are in considerably lower abundance relative to transcripts associated with the previous physiological state. Selective translation of transcripts by distinct ribosomes could form the basis for adaptive evolution to new environments through a modular regulation of the translational systems.

scholarly journals Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasuko Matsuki ◽  
Yoshitaka Matsuo ◽  
Yu Nakano ◽  
Shintaro Iwasaki ◽  
Hideyuki Yoko ◽  
...  
Keyword(s):  
Er Stress ◽  
Ribosomal Protein ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Independent Manner ◽  
Eif2α Phosphorylation ◽  
The Unfolded Protein Response

AbstracteIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

scholarly journals Ribosomal protein S7 ubiquitination during ER stress in yeast is associated with selective mRNA translation and stress outcome

2020 ◽  
Author(s):  
Yasuko Matsuki ◽  
Yoshitaka Matsuo ◽  
Yu Nakano ◽  
Shintaro Iwasaki ◽  
Hideyuki Yoko ◽  
...  
Keyword(s):  
Er Stress ◽  
Ribosomal Protein ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Independent Manner ◽  
Eif2α Phosphorylation ◽  
The Unfolded Protein Response

ABSTRACTeIF2α phosphorylation-mediated translational regulation is crucial for global translation repression by various stresses, including the unfolded protein response (UPR). However, translational control during UPR has not been demonstrated in yeast. This study investigated ribosome ubiquitination-mediated translational controls during UPR. Tunicamycin-induced ER stress enhanced the levels of ubiquitination of the ribosomal proteins uS10, uS3 and eS7. Not4-mediated monoubiquitination of eS7A was required for resistance to tunicamycin, whereas E3 ligase Hel2-mediated ubiquitination of uS10 was not. Ribosome profiling showed that the monoubiquitination of eS7A was crucial for translational regulation, including the upregulation of the spliced form of HAC1 (HAC1i) mRNA and the downregulation of Histidine triad NucleoTide-binding 1 (HNT1) mRNA. Downregulation of the deubiquitinating enzyme complex Upb3-Bre5 increased the levels of ubiquitinated eS7A during UPR in an Ire1-independent manner. These findings suggest that the monoubiquitination of ribosomal protein eS7A plays a crucial role in translational controls during the ER stress response in yeast.

scholarly journals Narrow Leaf21, Encoding Ribosomal Protein RPS3A, Controls Leaf Development in Rice

2021 ◽  
Author(s):  
Muhammad Uzair ◽  
Haixin Long ◽  
Syed Adeel Zafar ◽  
Suyash B Patil ◽  
Yan Chun ◽  
...  
Keyword(s):  
Leaf Development ◽  
Genomic Dna ◽  
Translational Regulation ◽  
Leaf Blade ◽  
Leaf Morphology ◽  
Vascular System ◽  
Small Subunit ◽  
Ribosome Profiling ◽  
Antibiotics Resistance ◽  
Leaf Phenotype

Abstract Leaf morphology influences photosynthesis, transpiration and ultimately crop yield. However, the molecular mechanism of leaf development is still not fully understood. Here, we identified and characterized the narrow leaf21 (nal21) mutant in rice (Oryza sativa), showing a significant reduction in leaf width, leaf length and plant height, and increased tiller number. Microscopic observation revealed defects in the vascular system and reduced epidermal cell size and number in the nal21 leaf blade. Map-based cloning revealed that NAL21 encodes a ribosomal small subunit protein RPS3A. Ribosome-targeting antibiotics resistance assay and ribosome profiling showed a significant reduction in the free 40S ribosome subunit in the nal21 mutant. The nal21 mutant showed aberrant auxin responses in which multiple auxin response factors (ARFs) harboring upstream open reading frames (uORFs) in their 5′-untranslated region (5’-UTR) were repressed at the translational level. The WUSCHEL-related homeobox 3A (OsWOX3A) gene, a key transcription factor involved in leaf blade lateral outgrowth, is also under the translational regulation by RPS3A. Transformation with modified OsARF11, OsARF16 and OsWOX3A genomic DNA lacking uORFs rescued the narrow leaf phenotype of nal21 to a better extent than transformation with their native genomic DNA, implying that RPS3A could regulate translation of ARFs and WOX3A through uORFs. Our results demonstrate that proper translational regulation of key factors involved in leaf development is essential to maintain normal leaf morphology.

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

Ribosome Structural Organization

1974 ◽  
Vol 32 ◽  
pp. 332-333
Author(s):  
James A. Lake
Keyword(s):  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Small Subunit ◽  
Structural Studies ◽  
X Ray Diffraction ◽  
Specific Antibodies ◽  
X Ray ◽  
E Coli ◽  
Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

Sign in / Sign up

Export Citation Format

Share Document