Avoidance of Long Mononucleotide Repeats in Codon Pair Usage

Protein is an essential component for life, and its synthesis is mediated by codons in any organisms on earth. While some codons encode the same amino acid, their usage is often highly biased. There are many factors that can cause the bias, but a potential effect of mononucleotide repeats, which are known to be highly mutable, on codon usage and codon pair preference is largely unknown. In this study we performed a genomic survey on the relationship between mononucleotide repeats and codon pair bias in 53 bacteria, 68 archaea, and 13 eukaryotes. By distinguishing the codon pair bias from the codon usage bias, four general patterns were revealed: strong avoidance of five or six mononucleotide repeats in codon pairs; lower observed/expected (o/e) ratio for codon pairs with C or G repeats (C/G pairs) than that with A or T repeats (A/T pairs); a negative correlation between genomic GC contents and the o/e ratios, particularly for C/G pairs; and avoidance of C/G pairs in highly conserved genes. These results support natural selection against long mononucleotide repeats, which could induce frameshift mutations in coding sequences. The fact that these patterns are found in all kingdoms of life suggests that this is a general phenomenon in living organisms. Thus, long mononucleotide repeats may play an important role in base composition and genetic stability of a gene and gene functions.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Temporal and Spatial Coexistence of Archaeal and Bacterial amoA Genes and Gene Transcripts in Lake Lucerne

Despite their crucial role in the nitrogen cycle, freshwater ecosystems are relatively rarely studied for active ammonia oxidizers (AO). This study of Lake Lucerne determined the abundance of both amoA genes and gene transcripts of ammonia-oxidizing archaea (AOA) and bacteria (AOB) over a period of 16 months, shedding more light on the role of both AO in a deep, alpine lake environment. At the surface, at 42 m water depth, and in the water layer immediately above the sediment, AOA generally outnumbered AOB. However, in the surface water during summer stratification, when both AO were low in abundance, AOB were more numerous than AOA. Temporal distribution patterns of AOA and AOB were comparable. Higher abundances of amoA gene transcripts were observed at the onset and end of summer stratification. In summer, archaeal amoA genes and transcripts correlated negatively with temperature and conductivity. Concentrations of ammonium and oxygen did not vary enough to explain the amoA gene and transcript dynamics. The observed herbivorous zooplankton may have caused a hidden flux of mineralized ammonium and a change in abundance of genes and transcripts. At the surface, AO might have been repressed during summer stratification due to nutrient limitation caused by active phytoplankton.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules (1). Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii (2). 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. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production (1). (1) Gebetsberger J. and Polacek N. (2013), RNA Biol. 10:1798-1808 (2) Gebetsberger J. et. al. (2012), Archaea, Article ID 260909

The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion

While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)-targeted DNA-deletion system. The first sRNA-targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference-derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

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.