Periodic Reporting for period 1 - bacRBP (Exploring the expanding universe of RNA-binding proteins in bacteria)
Reporting period: 2022-11-01 to 2025-04-30
All organisms including bacteria use diverse modes of cellular control as they cope with changing environments. RNA-protein complexes are central to these processes. While deep sequencing approaches have revealed a wealth of RNAs, the world of bacterial RNA-binding proteins (RBPs) is still largely uncharted. Typically, RBPs interact with their RNA targets via distinct RNA-binding domains (RBDa). However, a growing number of proteins that lack these domains seem to interact with RNA as well. In prokaryotes, such unconventional RBPs (ucRBPs) remain largely unexplored, in part because methods for global RNA interactome capture (RIC) in bacteria are missing. We developed a novel RIC approach for bacteria that relies on primary transcript capture (CoCAP). Our pilot study captured known RBPs but also uncovered numerous new RBP candidates pointing towards a wealth of unexplored RBPs involved in cellular control in bacteria.
My bacRBP project explores the identity and functional diversity of such novel RBPs in bacteria with a focus on ucRBPs that play crucial roles in cellular physiology. I will tackle this through three objectives (Fig. 1) leveraging two model bacteria (Salmonella and Campylobacter):
1) Elucidate bacterial primary RBPomes during stress- and infection-relevant conditions.
2) Identify mechanisms and cellular functions of two widely conserved KH-domain RBPs.
3) Determine how unconventional RBPs influence and are influenced by bound RNAs.
Our work will provide a broadly applicable method for primary RBPome capture, vastly expand the set of bacterial RBPs, and reveal new layers of cellular control by ucRBPs.
Figure 1: Objectives of bacRBP. Our overarching hypothesis is that a vast, unexplored universe of unconventional RBPs exists in bacteria that mediate key biological processes. In Objective 1, we aim to expand the set of known bacterial RBPs by applying CoCAP to Salmonella and C. jejuni as well as examine potential functions of selected transcriptional regulator RBP candidates. In Objective 2 we explore the cellular roles and underlying molecular mechanisms of the KH-domain proteins KhpA/B in C. jejuni. In Objective 3, we aim to find out whether unconventional RBPs affect bound RNAs or vice versa.
My bacRBP project explores the identity and functional diversity of such novel RBPs in bacteria with a focus on ucRBPs that play crucial roles in cellular physiology. I will tackle this through three objectives (Fig. 1) leveraging two model bacteria (Salmonella and Campylobacter):
1) Elucidate bacterial primary RBPomes during stress- and infection-relevant conditions.
2) Identify mechanisms and cellular functions of two widely conserved KH-domain RBPs.
3) Determine how unconventional RBPs influence and are influenced by bound RNAs.
Our work will provide a broadly applicable method for primary RBPome capture, vastly expand the set of bacterial RBPs, and reveal new layers of cellular control by ucRBPs.
Figure 1: Objectives of bacRBP. Our overarching hypothesis is that a vast, unexplored universe of unconventional RBPs exists in bacteria that mediate key biological processes. In Objective 1, we aim to expand the set of known bacterial RBPs by applying CoCAP to Salmonella and C. jejuni as well as examine potential functions of selected transcriptional regulator RBP candidates. In Objective 2 we explore the cellular roles and underlying molecular mechanisms of the KH-domain proteins KhpA/B in C. jejuni. In Objective 3, we aim to find out whether unconventional RBPs affect bound RNAs or vice versa.
Primary RBPomes of Salmonella and C. jejuni
Our pilot CoCAP screen of S. Typhimurium and C. jejuni successfully captured known RBPs such as Hfq and ProQ in Salmonella but also revealed diverse potential novel RBPs in both organisms. Functional enrichment and domain analyses in these datasets demonstrated the capacity and specificity of CoCAP to identify proteins that interact with RNA independent of the bacterial species. This included RBPs with distinct RNA-binding domains but also many unconventional RBP candidates without known RBDs. We tested the RNA-binding capacity of selected Salmonella ucRBP candidates via PNK assays and found that the majority is able to bind RNA in vivo. We are currently exploring their RNA targetomes via RIP- and CLIP-seq approaches.
KH-domain RBPs in C. jejuni
Compared to Salmonella, C. jejuni lacks homologs of canonical global RNA binders, such as Hfq and ProQ. In this Epsilonproteobacterium, CoCAP identified a pair of KH-domain RBPs (KhpA/B) that are widespread in bacteria and whose function is largely unexplored. We had confirmed that both RBPs interact with each other, and now addressed several questions including if each protein has a distinct set of RNA targets or if they only bind RNA when in a complex. To this end, we performed several RIP-seq experiments of KhpA and KhpB in the presence and absence of the respective other RBP. Our results suggest that both proteins bind a significant number of transcripts with both KhpA-/B-specific and -common RNA targetomes. A strong reduction of bound transcripts for both RBPs when their binding partner is absent indicates that their respective RNA-binding capacity depends on the presence of the other. By mutating individual domains of both KhpA/B, we also identified the domains responsible for binding RNA in both proteins. In addition, we are also exploring the protein interactome of KhpA/B for which we consolidated our preliminary mass spectrometry(MS)-based analysis with three more replicates. Our results suggest KhpB to be a major protein interactor and implicate the involvement of KhpA/B in RNA-related higher order protein complexes. Validation for selected candidate protein interaction partners is currently ongoing.
To identify cellular functions and molecular mechanisms of KhpA/B-based regulation we are currently exploring possible phenotypes affected by the absence of khpA/B and by their ability to bind RNA. This includes for example effects on C. jejuni growth as well as subcellular localization patterns of KhpA/B and their potential RNA/protein binding partners. These experiments are consolidated by target validations and follow-up studies of our RIP-seq and protein-interactome studies.
Our pilot CoCAP screen of S. Typhimurium and C. jejuni successfully captured known RBPs such as Hfq and ProQ in Salmonella but also revealed diverse potential novel RBPs in both organisms. Functional enrichment and domain analyses in these datasets demonstrated the capacity and specificity of CoCAP to identify proteins that interact with RNA independent of the bacterial species. This included RBPs with distinct RNA-binding domains but also many unconventional RBP candidates without known RBDs. We tested the RNA-binding capacity of selected Salmonella ucRBP candidates via PNK assays and found that the majority is able to bind RNA in vivo. We are currently exploring their RNA targetomes via RIP- and CLIP-seq approaches.
KH-domain RBPs in C. jejuni
Compared to Salmonella, C. jejuni lacks homologs of canonical global RNA binders, such as Hfq and ProQ. In this Epsilonproteobacterium, CoCAP identified a pair of KH-domain RBPs (KhpA/B) that are widespread in bacteria and whose function is largely unexplored. We had confirmed that both RBPs interact with each other, and now addressed several questions including if each protein has a distinct set of RNA targets or if they only bind RNA when in a complex. To this end, we performed several RIP-seq experiments of KhpA and KhpB in the presence and absence of the respective other RBP. Our results suggest that both proteins bind a significant number of transcripts with both KhpA-/B-specific and -common RNA targetomes. A strong reduction of bound transcripts for both RBPs when their binding partner is absent indicates that their respective RNA-binding capacity depends on the presence of the other. By mutating individual domains of both KhpA/B, we also identified the domains responsible for binding RNA in both proteins. In addition, we are also exploring the protein interactome of KhpA/B for which we consolidated our preliminary mass spectrometry(MS)-based analysis with three more replicates. Our results suggest KhpB to be a major protein interactor and implicate the involvement of KhpA/B in RNA-related higher order protein complexes. Validation for selected candidate protein interaction partners is currently ongoing.
To identify cellular functions and molecular mechanisms of KhpA/B-based regulation we are currently exploring possible phenotypes affected by the absence of khpA/B and by their ability to bind RNA. This includes for example effects on C. jejuni growth as well as subcellular localization patterns of KhpA/B and their potential RNA/protein binding partners. These experiments are consolidated by target validations and follow-up studies of our RIP-seq and protein-interactome studies.
Our bacRBP project is making progress in the characterization of both conventional and unconventional RBPs in C. jejuni and Salmonella. Our work will provide a broadly applicable method for RIC in bacteria, expand the repertoire of RBPs in prokaryotes, and reveal new layers of gene regulation in bacterial pathogens. Moreover, our results on the KH-domain proteins in C. jejuni have the potential to reveal their thus far still largely unclear cellular roles.