Biology subjects

Bandyra, K. J.

Publications and source records attributed to Bandyra, K. J..

6 recordsLinked to original sources

bioRxiv2024

Human FASTK preferentially binds single-stranded and G-rich RNA

Fas-activated serine/threonine kinase (FASTK) is the founding member of the FASTKD protein family, which was shown to regulate the fate of mRNA molecules on multiple levels. The mitochondrial variant of FASTK co-localizes with mitochondrial RNA granules and regulates degradation of mitochondrial mRNAs, whereas the cytoplasmic and nuclear forms of FAST are involved in regulation of alternative splicing, cytoplasmic RNA granule formation and mRNA translation. Despite these multiple roles of FASTK in mRNA biology, the exact rules of RNA recognition by this protein remained undetermined. Here, we demonstrate direct RNA binding by purified human FASTK and show its preference for single-stranded G-rich sites and RNA G-quadruplexes. Addition of FASTK alone was sufficient to achieve protection of mitochondrial mRNAs from degradation by the degradosome. Structural characterization by SAXS showed that FASTK in solution is a monomer with an extended conformation. Point mutagenesis studies supported the structural predictions of an exposed RNA-binding interface in the central helical region, preceded by a smaller, flexibly attached, helical N-terminal domain. We provide the first such extensive in vitro characterization of the RNA binding properties for a representative of the FASTKD protein family, and suggest how these intrinsic properties may underly FASTK function in mRNA metabolism.

molecular biology↗

bioRxiv2024

Depletion of essential GroEL protein in Escherichia coli using Clp-Interacting Peptidic Protein Erasers (CLIPPERs)

New, universal tools for targeted protein degradation in bacteria can help to accelerate protein function studies and antimicrobial research. We have developed a new method for degrading bacterial proteins using plasmid-encoded degrader peptides which deliver target proteins for degradation by a highly conserved ClpXP protease. We demonstrated the mode of action of the degraders on a challenging essential target, GroEL. The studies in bacteria were complemented by in vitro binding and structural studies. Expression of degrader peptides resulted in a temperature-dependent growth inhibition and depletion of GroEL levels over time. The reduction of GroEL levels was accompanied by dramatic proteome alterations. The presented method offers a new alternative approach for regulating protein levels in bacteria without genomic modifications or tag fusions. Our studies demonstrate that ClpXP is an attractive protease for the future use in bacterial targeted protein degradation.

molecular biology↗

bioRxiv2021

Escherichia coli ribosomal protein S1 enhances the kinetics of ribosome biogenesis and RNA decay

Escherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

microbiology↗

bioRxiv2021

RNase E endonuclease activity and its inhibition by pseudoridine

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzymes hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multi-component RNA degradosome assembly. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5{square} nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognised uracil is isomerised to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric auto-regulation. The catalytic power of the enzyme is boosted when it is assembled into the multi-enzyme RNA degradosome, most likely as a consequence of substrate channeling. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.

biochemistry↗

bioRxiv2020

Antisense oligonucleotides target a nearly invariant structural element from the SARS-CoV-2 genome and drive RNA degradation

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stemloop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3 UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 infection in cell culture. Our results thus permit us to suggest that the s2m element is a site of vulnerability readily targeted by ASOs, which show promise as anti-viral agents.

molecular biology↗

bioRxiv2018

Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3' UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

microbiology↗