cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

Gebetsberger, Jennifer; Fricker, Roger; Polacek, Norbert

doi:10.1007/978-1-4939-2547-6_13

Cited by 4 publications

(2 citation statements)

References 12 publications

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“…Ribosome-associated small RNAs were isolated as described 49 . The cDNA library was prepared using the TruSeq Small RNA Library Prep kit ( Illumina ) and sequenced using the Illumina HiSeq 2000 platform.…”

Section: Methodsmentioning

confidence: 99%

A tRNA half modulates translation as stress response in Trypanosoma brucei

et al. 2019

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In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.

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Section: Methodsmentioning

confidence: 99%

A tRNA half modulates translation as stress response in Trypanosoma brucei

et al. 2019

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“…cDNA library encoding ribosome-associated RNA in the size range between 20–500 nucleotides was prepared and deep-sequenced as described previously 12 , 15 , 32 . The obtained sequence reads have been deposited in ENA (Acc.…”

Section: Methodsmentioning

confidence: 99%

mRNA-specific translation regulation by a ribosome-associated ncRNA in Haloferax volcanii

Wyss

Waser

Gebetsberger

et al. 2018

Sci Rep

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Regulation of gene expression at the translational level allows rapid adaptation of cellular proteomes to quickly changing environmental conditions and is thus central for prokaryotic organisms. Small non-coding RNAs (sRNAs) have been reported to effectively orchestrate translation control in bacteria and archaea mainly by targeting mRNAs by partial base complementarity. Here we report an unprecedented mechanism how sRNAs are capable of modulating protein biosynthesis in the halophilic archaeon Haloferax volcanii. By analyzing the ribosome-associated ncRNAs (rancRNAs) under different stress conditions we identified an intergenic sRNA, termed rancRNA_s194, that is primarily expressed during exponential growth under all tested conditions. By interaction with the ribosome rancRNA_s194 inhibits peptide bond formation and protein synthesis in vitro but appears to target a specific mRNA in vivo. The respective knock-out strain shows a reduced lag phase in media containing xylose as sole carbon source and outcompetes the wildtype cells under these conditions. Mass spectrometry, polysome profiling and mRNA binding competition experiments suggest that rancRNA_s194 prevents the cstA mRNA from being efficiently translated by H. volcanii ribosomes. These findings enlarge the regulatory repertoire of archaeal sRNAs in modulating post-transcriptional gene expression.

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Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli

Luidalepp

Berger

Joss

et al. 2016

Journal of Molecular Biology

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Stationary-phase bacterial cells are characterized by vastly reduced metabolic activities yielding a dormant-like phenotype. Several hibernation programs ensure the establishment and maintenance of this resting growth state. Some of the stationary phase-specific modulations affect the ribosome and its translational activity directly. In stationary-phase Escherichia coli, we observed the appearance of a 16S rRNA fragmentation event at the tip of helix 6 within the small ribosomal subunit (30S). Stationary-phase 30S subunits showed markedly reduced activities in protein biosynthesis. On the other hand, the functional performance of stationary-phase large ribosomal subunits (50S) was indistinguishable from particles isolated from exponentially growing cells. Introduction of the 16S rRNA cut in vitro at helix 6 of exponential phase 30S subunits renders them less efficient in protein biosynthesis. This indicates that the helix 6 fragmentation is necessary and sufficient to attenuate translational activities of 30S ribosomal subunits. These results suggest that stationary phase-specific cleavage of 16S rRNA within the 30S subunit is an efficient means to reduce global translation activities under non-proliferating growth conditions.

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cDNA Library Generation for the Analysis of Small RNAs by High-Throughput Sequencing

Cited by 4 publications

References 12 publications

A tRNA half modulates translation as stress response in Trypanosoma brucei

A tRNA half modulates translation as stress response in Trypanosoma brucei

mRNA-specific translation regulation by a ribosome-associated ncRNA in Haloferax volcanii

Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli

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