Genomic knock-out of rancRNAs in the archaeon H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms [1]. Herein included is the prominent example of gene silencing caused by micro RNAs (miRNAs) and small interfering RNAs (siRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of RNAs among the well-studied ncRNAs that target the ribosome itself [2,3]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. Recent studies show the presence of small regulatory RNAs (sRNAs) in archaea which are involved in many biological processes including stress response and metabolic regulation [4]. To date the biological function and the targets of these archaeal sRNAs are only described for a few examples. There are reports of sRNAs binding to the 5’ as well as to the 3’ of mRNAs [5,6]. In addition to these findings, a tRNA derived fragment (tRF) of Valine tRNA was found in a genomic screen of RNAs associated with the ribosome in H. volcanii in our laboratory [3]. This Valine tRF seems to be processed in a stress-dependent manner and showed in vitro binding to the ribosome and inhibited in vitro translation. These results showed that Valine tRF is capable to regulate translation in H. volcanii by targeting the ribosome. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression patterns in response to stress conditions. To investigate the biological relevance of some of the ribosome-associated ncRNA candidates, knock-out and phenotypic characterization studies are done. The genomic knock out of a hypothetical ORF (198nt), where one putative rancRNA candidate (46nt) named IG33 was detected in the library at the beginning of the ORF, showed interesting growth phenotype under specific stress conditions. Furthermore a strain with an introduced start to stop codon mutation in this hypothetical ORF still shows the same phenotype indicating that rather the missing protein than the missing sRNA causes this growth phenotype.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

Biology of tRNA halves in Trypanosoma brucei

Although T. brucei has to challenge tremendous environment changes, e.g. switch from the bloodstream form in mammalian hosts to the mid gut form present in tsetse flies, there is no evidence for differential regulation of RNA Pol II transcription. Instead, constitutive transcription appears to occur. This observation indicates that protein levels have to be regulated by post-transcriptional mechanisms. It has been shown that non-protein coding RNAs (ncRNAs) are crucial in regulatory networks (e.g. chromosome remodelling; RNA polymerase activity; mRNA turnover; etc.), but all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. This is unexpected, since the ribosome has a central role during gene expression and due to the assumption that the primordial translation system most likely received direct regulatory input from small molecules including ncRNA cofactors. In our lab, it has been discovered that ncRNAs are able to directly bind to the ribosome, therefore influencing the translation rate in Haloferax volcanii and Saccharomyces cerevisiae. In order to extend this idea of ribosome-binding ncRNAs in mammalian parasites, we want to investigate this mechanism in T. brucei. Accordingly, we performed a genomic screen for small ribosome-associated RNAs followed by functional analyses of possible candidates. With the help of this genomic screen, we found tRNAs that are alternated and tRNA halves that are differentially expressed upon nutritional stress.

An intergenic non-coding RNA targets and regulates the ribosome in H. volcanii.

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the nonprotein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms (e.g. gene silencing by microRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of ncRNAs, which target the ribosome itself [Gebetsberger et al., 2012/ Pircher et al, 2014]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression of rancRNAs during different growth phases or under specific stress conditions. To investigate the biological relevance of these rancRNAs, knock-outs were generated in H. volcanii which were used for phenotypic characterization studies. The rancRNA s194 showed association with the 50S ribosomal subunit in vitro and in vivo and was capable of inhibiting peptide bond formation. These preliminary data for the rancRNA s194 make it an interesting candidate for further functional studies to identify the molecular mechanisms by which rancRNAs can modulate protein biosynthesis. Characterization of further rancRNA candidates are also underway.

A comparative study on manual and automatic slice-to-volume registration of CT images

In order to assess the clinical relevance of a slice-to-volume registration algorithm, this technique was compared to manual registration. Reformatted images obtained from a diagnostic CT examination of the lower abdomen were reviewed and manually registered by 41 individuals. The results were refined by the algorithm. Furthermore, a fully automatic registration of the single slices to the whole CT examination, without manual initialization, was also performed. The manual registration error for rotation and translation was found to be 2.7+/-2.8 degrees and 4.0+/-2.5 mm. The automated registration algorithm significantly reduced the registration error to 1.6+/-2.6 degrees and 1.3+/-1.6 mm (p = 0.01). In 3 of 41 (7.3%) registration cases, the automated registration algorithm failed completely. On average, the time required for manual registration was 213+/-197 s; automatic registration took 82+/-15 s. Registration was also performed without any human interaction. The resulting registration error of the algorithm without manual pre-registration was found to be 2.9+/-2.9 degrees and 1.1+/-0.2 mm. Here, a registration took 91+/-6 s, on average. Overall, the automated registration algorithm improved the accuracy of manual registration by 59% in rotation and 325% in translation. The absolute values are well within a clinically relevant range.

When small non-coding RNAs meet the ribosome: Tuning the translational machinery

The functions of ribosomes in translation are complex and involve different types of activities critical for decoding the genetic code, linkage of amino acids via amide bonds to form polypeptide chains, as well as the release and proper targeting of the synthesized protein. Non-protein-coding RNAs (ncRNAs) have been recognized to be crucial in establishing regulatory networks.1 However all of the recently discovered ncRNAs involved in translation regulation target the mRNA rather than the ribosome. The main goal of this project is to identify potential novel ncRNAs that directly bind and possibly regulate the ribosome during protein biosynthesis. To address this question we applied various stress conditions to the archaeal model organism Haloferax volcanii and deep-sequenced the ribosome-associated small ncRNA interactome. In total we identified 6.250 ncRNA candidates. Significantly, we observed the emersed presence of tRNA-derived fragments (tRFs). These tRFs have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNAs. Here we present evidence that tRFs from H. volcanii directly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome a 26 residue long fragment originating from the 5’ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production.2 Currently we are investigating the binding site of this tRF on the 30S subunit in more detail.

Eukaryotic Initiation Factor 4G Suppresses Nonsense-Mediated mRNA Decay by Two Genetically Separable Mechanisms

Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

How and where are nonsense mRNAs degraded in mammalian cells?

The nonsense-mediated mRNA decay (NMD) pathway is responsible for the rapid degradation of eukaryotic mRNAs on which ribosomes fail to terminate translation properly. NMD thereby contributes to the elimination of aberrant mRNAs, improving the fidelity of gene expression, but also serves to regulate gene expression at the post-transcriptional level. Here we discuss recent evidence as to how and where mRNAs targeted to NMD are degraded in human cells. We discuss accumulating evidence that the decay step of human NMD can be initiated by two different mechanisms: either by SMG6-mediated endonucleolytic cleavage near the aberrant stop codon, or by deadenylation and decapping. While there is evidence that mRNAs targeted for NMD have the capacity to accumulate with other translationally repressed mRNAs in P-bodies, there is currently no evidence that this is required for the degradation of the NMD substrate. It therefore remains an open question whether NMD in human cells is restricted to a particular cellular location or whether it can be initiated wherever translation of the NMD substrate takes place

Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML

Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress-associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).