Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress

AbstractSynthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.

Download Full-text

Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8

Genome Biology ◽

10.1186/s13059-021-02345-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Audrey Montigny ◽

Patrizia Tavormina ◽

Carine Duboe ◽

Hélène San Clémente ◽

Marielle Aguilar ◽

...

Keyword(s):

Regulatory Peptides ◽

Ribosome Profiling ◽

Small Peptides ◽

Molecular Approaches ◽

Genome Expression ◽

Genome Wide ◽

Short Orfs ◽

Regulatory Potential ◽

Regulatory Feedback Loop ◽

And Function

Abstract Background Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. Results We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. Conclusion Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

Download Full-text

Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽

10.3390/biomedicines9080911 ◽

2021 ◽

Vol 9 (8) ◽

pp. 911

Author(s):

Joana Silva ◽

Pedro Nina ◽

Luísa Romão

Keyword(s):

Translational Regulation ◽

Regulatory Elements ◽

Ribosome Profiling ◽

Leader Sequence ◽

Open Reading Frames ◽

Regulatory Function ◽

Coding Sequence ◽

Abc Protein ◽

Upstream Open Reading Frames ◽

Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

Download Full-text

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

Nature Communications ◽

10.1038/s41467-021-25411-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Puneet Sharma ◽

Jie Wu ◽

Benedikt S. Nilges ◽

Sebastian A. Leidel

Keyword(s):

Human Cells ◽

Ribosome Profiling ◽

Model Organisms ◽

Treatment Conditions ◽

Genome Wide ◽

Optimized Protocol ◽

Gene Level ◽

Translation Inhibitor ◽

Species Specific ◽

Conformational Restrictions

AbstractRibosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

Download Full-text

Genome-wide study of aging and oxidative stress response in Drosophilamelanogaster

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.260496697 ◽

2000 ◽

Vol 97 (25) ◽

pp. 13726-13731 ◽

Cited By ~ 216

Author(s):

S. Zou ◽

S. Meadows ◽

L. Sharp ◽

L. Y. Jan ◽

Y. N. Jan

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Oxidative Stress Response ◽

Genome Wide ◽

And Oxidative Stress ◽

Genome Wide Study

Download Full-text