hGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.

Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and crossregulation. They are highly abundant and many localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a "one-fits-all" plasmid, and integration into any cell line with endogenously GFP-tagged proteins allows for an inducible, rapid, and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2, and NONO are completely cleared from nuclear speckles and paraspeckles within 2 h. hGRAD works in various cell types, is more efficient than previous methods, and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered transient transcript changes, compensatory mechanisms, and an effect of SRSF5 on transcript stability.

Thinking Outside the Frame: Impacting Genomes Capacity by Programmed Ribosomal Frameshifting.

Translation facilitates the transfer of the genetic information stored in the genome via messenger RNAs to a functional protein and is therefore one of the most fundamental cellular processes. Programmed ribosomal frameshifting is a ubiquitous alternative translation event that is extensively used by viruses to regulate gene expression from overlapping open reading frames in a controlled manner. Recent technical advances in the translation field enabled the identification of precise mechanisms as to how and when ribosomes change the reading frame on mRNAs containing cis-acting signals. Several studies began also to illustrate that trans-acting RNA modulators can adjust the timing and efficiency of frameshifting illuminating that frameshifting can be a dynamically regulated process in cells. Here, we intend to summarize these new findings and emphasize how it fits in our current understanding of PRF mechanisms as previously described.

The Ribosomal Protein L5 Functions During Xenopus Anterior Development Through Apoptotic Pathways.

Ribosomal biogenesis is a fundamental process necessary for cell growth and division. Ribosomal protein L5 (Rpl5) is part of the large ribosomal subunit. Mutations in this protein have been associated with the congenital disease Diamond Blackfan anemia (DBA), a so called ribosomopathy. Despite of the ubiquitous need of ribosomes, clinical manifestations of DBA include tissue-specific symptoms, e.g., craniofacial malformations, eye abnormalities, skin pigmentation failure, cardiac defects or liver cirrhosis. Here, we made use of the vertebrate model organism Xenopus laevis and showed a specific expression of rpl5 in the developing anterior tissue correlating with tissues affected in ribosomopathies. Upon Rpl5 knockdown using an antisense-based morpholino oligonucleotide approach, we showed different phenotypes affecting anterior tissue, i.e., defective cranial cartilage, malformed eyes, and microcephaly. Hence, the observed phenotypes in Xenopus laevis resemble the clinical manifestations of DBA. Analyses of the underlying molecular basis revealed that the expression of several marker genes of neural crest, eye, and brain are decreased during induction and differentiation of the respective tissue. Furthermore, Rpl5 knockdown led to decreased cell proliferation and increased cell apoptosis during early embryogenesis. Investigating the molecular mechanisms underlying Rpl5 function revealed a more than additive effect between either loss of function of Rpl5 and loss of function of c-Myc or loss of function of Rpl5 and gain of function of Tp53, suggesting a common signaling pathway of these proteins. The co-injection of the apoptosis blocking molecule Bcl2 resulted in a partial rescue of the eye phenotype, supporting the hypothesis that apoptosis is one main reason for the phenotypes occurring upon Rpl5 knockdown. With this study, we are able to shed more light on the still poorly understood molecular background of ribosomopathies.