Biology subjects

Steglich, C.

Publications and source records attributed to Steglich, C..

4 recordsLinked to original sources

bioRxiv2022

Feedback regulation of RNase E during UV-stress response in the cyanobacterium Synechocystis sp. PCC 6803

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5'-untranslated region (5' UTR); however, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation via its own 5' UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

microbiology↗

bioRxiv2021

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3 end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

microbiology↗

bioRxiv2021

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the {Delta}crhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets. HighlightThe cyanobacterial DEAD-box RNA helicase CrhR binds mainly photosynthesis-associated and redox-controlled transcripts connecting its regulation, localization and phenotypes of mutants for the first time with a set of potential RNA targets.

plant biology↗

bioRxiv2020

Discovery of a novel small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded. This degradation is tightly coordinated, follows a genetic program and involves small proteins serving as proteolysis adaptors. Here, we describe the role of NblD, a novel factor in this process in cyanobacteria. NblD is a cysteine-rich, 66-amino acid small protein that becomes rapidly induced upon nitrogen starvation. Deletion of the nblD gene in the cyanobacterium Synechocystis prevents the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype. Competition experiments provided direct evidence for the physiological importance of NblD. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type. Overexpression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. Transcriptome analysis revealed that nitrogen starvation correctly induces nblA1/2 transcription in the {Delta}nblD strain implying that NblD does not act as a transcriptional (co-)regulator. However, fractionation and coimmunoprecipitation experiments indicated the presence of NblD in the phycobilisome fraction and identified the {beta}-phycocyanin subunit as its target. These data add NblD as a new factor to the genetically programmed response to nitrogen starvation and demonstrate that it plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. Significance StatementDuring genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the small protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells run low in nitrogen, a condition that frequently occurs in nature. Similar to the NblA proteins that label phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration.

microbiology↗