State-of-the-art and preparatory work

Ribonucleases (RNases) are required for RNA maturation from precursor molecules and turn‐over of mRNA in all living organisms. While mechanisms of RNA processing and degradation have been intensively studied in eukarya and bacteria these processes are much less understood in archaea. Our work revealed the presence of a multi‐protein complex in the hyperthermophilic archaeon Sulfolobus solfataricus, which shares many features with the eukaryotic exosome but also shows different composition and activities (1,2). The archaeal exosome can be reconstituted in vitro from recombinant subunits, exhibits RNA degrading as well as polymerization activity (3, 4, 5) and is associated with the membrane (6).

It emerged over the last ten years that small non‐coding RNAs (sRNAs) have important regulatory functions in prokaryotes. Ribonucleases have a crucial role in RNA‐mediated regulation since they take part in maturation of sRNAs and in their turn‐over. Pairing of an sRNA to its target mRNA can initiate degradation of both by double‐strand specific RNases. The RNA chaperone Hfq often assists in RNA hybrid formation and can influence the stability of the RNAs. Recently we identified sRNAs in the facultatively photosynthetic bacterium Rhodobacter sphaeroides. Interestingly one of these sRNAs is processed only in the presence of singlet oxygen (7). Another sRNA is involved in regulation of photosynthesis gene expression and its processing varies with changes in oxygen tension.

References

1. Evguenieva‐Hackenberg, E., Walter, P., Hochleitner, E., Lottspeich, F. & Klug, G. (2003). An exosome‐like complex in Sulfolobus solfataricus. EMBO rep 4, 889‐893.

2. Lorentzen, E., Walter, P., Fribourg, S., Evguenieva‐Hackenberg, E., Klug, G. & Conti, E. (2005) The archaeal exosome core is a hexameric ring structure with three catalytic subunits. Nature Struct Mol Biol 12, 575‐581.

3. Portnoy, V., Evguenieva‐Hackenberg, E., Klein, F., Walter, P., Lorentzen, E., Klug, G. & Schuster, G. (2005) RNA polyadenylation in Archaea: not observed in Haloferax while the exosome polynucleotidylates RNA in Sulfolobus. EMBO rep 6,1188‐1193.

4. Walter, P., Klein, F., Lorentzen, E., Ilchmann, A., Klug, G. & Evguenieva‐Hackenberg, E. (2006) Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus. Mol Microbiol 62, 1076‐1089.

5. Evguenieva‐Hackenberg, E., Roppelt, V., Finsterseifer, P. & Klug, G. (2008) Rrp4 and Csl4 are needed for efficient degradation but not for polyadenylation of synthetic and natural RNA by the archaeal exosome. Biochemistry 47, 13158‐13168.

6. Roppelt, V., Hobel, C. F. V., Albers, S. V., Lassek, C., Klug, G. & Evguenieva‐Hackenberg, E. (2010) FEBS Lett. in revision

7. Berghoff, B. A., Glaeser, J., Sharma, C., Vogel, J. & Klug, G. (2009) Photooxidative stress‐induced and abundant small RNAs in Rhodobacter sphaeroides. Mol Microbiol 74, 1497‐1512.

Aims

The aims of this project are

I. the elucidation of the in vivo function and molecular basis of substrate specificity of the archaeal exosome.

II. to unravel how external stimuli can specifically affect processing of bacterial sRNAs.

Work programme and methods

Using in vivo expressed tagged subunits of the exosome, large scale purification of the complex directly from S. solfataricus will be established, enabling the in vitro analysis of subunits with unknown function, the detection of further interaction partners of the exosome and structural studies on the native exosome. Another major goal of the project is to further analyse the mechanisms for substrate selection, processing and/or degradation by the exosome. The contribution of protein determinants (the impact of the RNA binding subunits Rrp4 and Csl4) to the interaction of the exosome with its substrates will be studied in detail. To this end, Rrp4 and Csl4 will be mutagenised and used for reconstitution of protein complexes which will be tested with suitably modified in vitro transcripts, based on native rRNA and mRNA substrates of the exosome.

We observed for the first time that bacterial sRNAs are processed in response to external stimuli. In the case of one of these sRNAs from R. sphaeroides the structure of the processing site is highly conserved among sRNA homologs found in other bacteria. The second sRNA is specific for R. sphaeroides. We will elucidate which RNases are involved in the observed processing by expressing the sRNAs in different mutant strains of Rhodobacter and E. coli. If the in vivo data point to an important role of known RNAses such as e.g. RNase E, RNase III or RNase J, we will also perform in vitro experiments with isolated proteins and in vitro transcripts. sRNAs with altered sequence and structure will be used for these in vitro studies and will also be expressed for in vivo studies to identify structural and/or sequence features leading to redox‐dependent processing.

Titles for dissertations (prospective)

• Contribution of exosomal subunits to substrate specificity in S. solfataricus

• Mechanisms of singlet oxygen‐dependent processing of RSs0682 in R. sphaeroides

• Mechanisms of oxgen‐dependent processing of RSs2430 in R. sphaeroides

Relationships/ connections within the research training group

Hartmann, Bindereif, Niepmann: RNA processing enzymes and mechanisms

Hartmann: RNA expression analysis of R. sphaeroides under conditions of copper limit/excess

Pingoud: catalytic mechanisms

Bindereif, Niepmann, Friedhoff, Shatsky: analysis of protein complexes

Friedhoff: Role of R. sphaeroides MMR in oxidative damage signalling

Wilde, Hartmann: processing of sRNAs in bacteria.

Bujnicki, Friedhoff, Marz: bioinformatics

Benefits of the scientific exchange

The IRTG has significantly intensified the exchange of methods and know‐how between the RNA groups and also made methodical expertise of other groups available for our projects. Of great benefit was also the bioinformatic expertise that the students were able to acquire.