Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome, characterized as a rare congenital bone marrow erythroid hypoplasia (OMIM#105650). Erythroid defect in DBA results in erythroblastopenia in bone marrow as a consequence of maturation blockade between the burst forming unit–erythroid and colony forming unit–erythroid developmental stages, leading to moderate to severe usually macrocytic aregenerative (<20 × 109/L of reticulocytes) anemia. Congenital malformations localized mostly in the cephalic area and in the extremities (thumbs), as well as short stature and cardiac and urogenital tract abnormalities, are a feature of 50% of the DBA-affected patients. A significant increased risk for malignancy has been reported. DBA is due to a defect in the ribosomal RNA (rRNA) maturation as a consequence of a heterozygous mutation in 1 of the 20 ribosomal protein genes. Besides classical DBA, some DBA-like diseases have been identified. The relation between the defect in rRNA maturation and the erythroid defect in DBA has yet to be fully defined. However, recent studies have identified a role for GATA1 either due to a specific defect in its translation or due to its defective regulation by its chaperone HSP70. In addition, excess free heme-induced reactive oxygen species and apoptosis have been implicated in the DBA erythroid phenotype. Current treatment options are either regular transfusions with appropriate iron chelation or treatment with corticosteroids starting at 1 year of age. The only curative treatment for the anemia of DBA to date is bone marrow transplantation. Use of gene therapy as a therapeutic strategy is currently being explored.

Dynamics and thermal sensitivity of ribosomal RNA maturation paths in plants

Ribosome biogenesis is a constitutive fundamental process for cellular function. Its rate of production depends on the rate of maturation of precursor ribosomal RNA (pre-rRNA). The rRNA maturation paths are marked by four dominant rate-limiting intermediates with cell-type variation of the processivity rate. We have identified that high temperature stress in plants, while halting the existing pre-rRNA maturation schemes, also transiently triggers an atypical pathway for 35S pre-rRNA processing. This pathway leads to production of an aberrant precursor rRNA, reminiscent of yeast 24S, encompassing 18S and 5.8S rRNA that do not normally co-occur together at sub-unit levels; this response is elicited specifically by high and not low temperatures. We show this response to be conserved in two other model crop plant species (Rice and Tomato). This pathway persists even after returning to normal growth conditions for 1 hour and is reset between 1-6 hours after stress treatment, likely, due to resumption of normal 35S pre-rRNA synthesis and processing. The heat-induced ITS2 cleavage-derived precursors and stalled P-A2-like precursors were heterogeneous in nature with a fraction containing polymeric (A) tails. Furthermore, high temperature treatment and subsequent fractionation resulted in polysome and precursor rRNA depletion.

Effects of Hydrogen Peroxide Stress on the Nucleolar Redox Environment and Pre-rRNA Maturation

Identifying biologically relevant molecular targets of oxidative stress may provide new insights into disease mechanisms and accelerate development of novel biomarkers. Ribosome biogenesis is a fundamental prerequisite for cellular protein synthesis, but how oxidative stress affects ribosome biogenesis has not been clearly established. To monitor and control the redox environment of ribosome biogenesis, we targeted a redox-sensitive roGFP reporter and catalase, a highly efficient H2O2 scavenger, to the nucleolus, the primary site for transcription and processing of rRNA in eukaryotic cells. Imaging of mouse 3T3 cells exposed to non-cytotoxic H2O2 concentrations revealed increased oxidation of the nucleolar environment accompanied by a detectable increase in the oxidative damage marker 8-oxo-G in nucleolar RNA. Analysis of pre-rRNA processing showed a complex pattern of alterations in pre-rRNA maturation in the presence of H2O2, including inhibition of the transcription and processing of the primary 47S transcript, accumulation of 18S precursors, and inefficient 3′-end processing of 5.8S rRNA. This work introduces new tools for studies of the redox biology of the mammalian nucleolus and identifies pre-rRNA maturation steps sensitive to H2O2 stress.

Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation

Ribosome biogenesis is among the founding processes in the cell. During the first stages of ribosome biogenesis, polycistronic precursor of ribosomal RNA passes complex multistage maturation after transcription. Quality control of preribosomal RNA (pre-rRNA) processing is precisely regulated by non-ribosomal proteins and structural features of pre-rRNA molecules, including modified nucleotides. However, many participants of rRNA maturation are still unknown or poorly characterized. We report that RNA m6A methyltransferase Mettl3 interacts with the 5′ external transcribed spacer (5′ETS) of the 47S rRNA precursor and modifies adenosine 196. We demonstrated that Mettl3 knockdown results in the increase of pre-rRNA processing rates, while intracellular amounts of rRNA processing machinery components (U3, U8, U13, U14, and U17 small nucleolar RNA (snoRNA)and fibrillarin, nucleolin, Xrn2, and rrp9 proteins), rRNA degradation rates, and total amount of mature rRNA in the cell stay unchanged. Increased efficacy of pre-rRNA cleavage at A’ and A0 positions led to the decrease of 47S and 45S pre-rRNAs in the cell and increase of mature rRNA amount in the cytoplasm. The newly identified conserved motif DRACH sequence modified by Mettl3 in the 5′-ETS region is found and conserved only in primates, which may suggest participation of m6A196 in quality control of pre-rRNA processing at initial stages demanded by increased complexity of ribosome biogenesis.

Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

The prokaryotic transcriptome is shaped by transcriptional and posttranscriptional events that define the characteristics of an RNA, including transcript boundaries, the base modification status, and processing pathways to yield mature RNAs. Currently, a combination of several specialised short-read sequencing approaches and additional biochemical experiments are required to describe all transcriptomic features. In this study, we present native RNA sequencing of bacterial (E. coli) and archaeal (H. volcanii, P. furiosus) transcriptomes employing the Oxford Nanopore sequencing technology. Based on this approach, we could address multiple transcriptomic characteristics simultaneously with single-molecule resolution. Taking advantage of long RNA reads provided by the Nanopore platform, we could (re-)annotate large transcriptional units and boundaries. Our analysis of transcription termination sites suggests that diverse termination mechanisms are in place in archaea. Moreover, we shed additional light on the poorly understood rRNA processing pathway in Archaea. One of the key features of native RNA sequencing is that RNA modifications are retained. We could confirm this ability by analysing the well-known KsgA-dependent methylation sites and mapping of N4-acetylcytosines modifications in rRNAs. Notably, we were able to follow the relative timely order of the installation of these modifications in the rRNA processing pathway.