Michal Malecki

The critical role of RNA processing and degradation in the control of gene expression

The continuous degradation and synthesis of prokaryotic mRNAs not only give rise to the metabolic changes that are required as cells grow and divide but also rapid adaptation to new environmental conditions. In bacteria, RNAs can be degraded by mechanisms that act independently, but in parallel, and that target different sites with different efficiencies. The accessibility of sites for degradation depends on several factors, including RNA higher-order structure, protection by translating ribosomes and polyadenylation status. Furthermore, RNA degradation mechanisms have shown to be determinant for the post-transcriptional control of gene expression. RNases mediate the processing, decay and quality control of RNA. RNases can be divided into endonucleases that cleave the RNA internally or exonucleases that cleave the RNA from one of the extremities. Just in Escherichia coli there are >20 different RNases. RNase E is a single-strand-specific endonuclease critical for mRNA decay in E. coli. The enzyme interacts with the exonuclease polynucleotide phosphorylase (PNPase), enolase and RNA helicase B (RhlB) to form the degradosome. However, in Bacillus subtilis, this enzyme is absent, but it has other main endonucleases such as RNase J1 and RNase III. RNase III cleaves double-stranded RNA and family members are involved in RNA interference in eukaryotes. RNase II family members are ubiquitous exonucleases, and in eukaryotes, they can act as the catalytic subunit of the exosome. RNases act in different pathways to execute the maturation of rRNAs and tRNAs, and intervene in the decay of many different mRNAs and small noncoding RNAs. In general, RNases act as a global regulatory network extremely important for the regulation of RNA levels.

The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway

The final step of cytoplasmic mRNA degradation proceeds in either a 5′‐3′ direction catalysed by Xrn1 or in a 3′‐5′ direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P‐bodies. Deletion of dis3l2+ is synthetically lethal with xrn1Δ, while deletion of dis3l2+ in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3′‐5′ RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

Bacterial adaptation to cold.

Micro-organisms react to a rapid temperature downshift by triggering a physiological response to ensure survival in unfavourable conditions. Adaptation includes changes in membrane composition and in the translation and transcription machineries. The cold shock response leads to a growth block and overall repression of translation; however, there is the induction of a set of specific proteins that help to tune cell metabolism and readjust it to the new conditions. For a mesophile like E. coli, the adaptation process takes about 4 h. Although the bacterial cold shock response was discovered over two decades ago we are still far from understanding this process. In this review, we aim to describe current knowledge, focusing on the functions of RNA-interacting proteins and RNases involved in cold shock adaptation.

RNA Degradation in Yeast and Human Mitochondria

Expression of mitochondrially encoded genes must be finely tuned according to the cells requirements. Since yeast and human mitochondria have limited possibilities to regulate gene expression by altering the transcription initiation rate, posttranscriptional processes, including RNA degradation, are of great importance. In both organisms mitochondrial RNA degradation seems to be mostly depending on the RNA helicase Suv3. Yeast Suv3 functions in cooperation with Dss1 ribonuclease by forming a two-subunit complex called the mitochondrial degradosome. The human ortholog of Suv3 (hSuv3, hSuv3p, SUPV3L1) is also indispensable for mitochondrial RNA decay but its ribonucleolytic partner has so far escaped identification. In this review we summarize the current knowledge about RNA degradation in human and yeast mitochondria. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

DMR1 ( CCM1/ YGR150C) of Saccharomyces cerevisiae Encodes an RNA-Binding Protein from the Pentatricopeptide Repeat Family Required for the Maintenance of the Mitochondrial 15S Ribosomal RNA

Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to ρ−/ρ0 petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Δ mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.

The RNase II/RNB family of exoribonucleases: putting the ‘Dis’ in disease

Important findings over the last years have shed new light onto the mechanistic details of RNA degradation by members of the RNase II/RNB family of exoribonucleases. Members of this family have been shown to be involved in growth, normal chloroplast biogenesis, mitotic control and cancer. Recently, different publications have linked human orthologs (Dis3 and Dis3L2) to important human diseases. This article describes the structural and biochemical characteristics of members of this family of enzymes, and the physiological implications that relate them with disease. WIREs RNA 2013, 4:607–615. doi: 10.1002/wrna.1180

Characterization of the RNase R association with ribosomes

BackgroundIn this study we employed the TAP tag purification method coupled with mass spectrometry analysis to identify proteins that co-purify with Escherichia coli RNase R during exponential growth and after temperature downshift.ResultsOur initial results suggested that RNase R can interact with bacterial ribosomes. We subsequently confirmed this result using sucrose gradient ribosome profiling joined with western blot analysis. We found that RNase R co-migrates with the single 30S ribosomal subunits. Independent data involving RNase R in the rRNA quality control process allowed us to hypothesize that the RNase R connection with ribosomes has an important physiological role.ConclusionsThis study leads us to conclude that RNase R can interact with ribosomal proteins and that this interaction may be a result of this enzyme involvement in the ribosome quality control.