Jan Medenbach

Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110

The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease.

RNA‐Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5′ splice‐site recognition is essential for both constitutive and regulated pre‐mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)‐specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C‐deficient U1 snRNPs. Surprisingly, genomewide RNA‐Seq analysis of mutant versus wild‐type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA‐mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C‐dependent 5′ splice sites uncovered an association with downstream intronic U‐rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.

Human U4/U6 snRNP Recycling Factor p110: Mutational Analysis Reveals the Function of the Tetratricopeptide Repeat Domain in Recycling

ABSTRACT After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions.

CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component.

The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions

Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

Human initiation factor eIF3 subunit b interacts with HCV IRES RNA through its N-terminal RNA recognition motif

Many viral mRNAs contain a 5′‐UTR RNA element called internal ribosome‐entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap‐independent translation. The IRES of hepatitis‐C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap‐dependent and cap‐independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N‐terminal‐RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed.

Promiscuity in post-transcriptional control of gene expression: Drosophila sex-lethal and its regulatory partnerships

The Drosophila RNA‐binding protein Sex‐lethal (Sxl) is a potent post‐transcriptional regulator of gene expression that controls female development. It regulates the expression of key factors involved in sex‐specific differences in morphology, behavior, and dosage compensation. Functional Sxl protein is only expressed in female flies, where it binds to U‐rich RNA motifs present in its target mRNAs to regulate their fate. Sxl is a very versatile regulator that, by shuttling between the nucleus and the cytoplasm, can regulate almost all aspects of post‐transcriptional gene expression including RNA processing, nuclear export, and translation. For these functions, Sxl employs multiple interactions to either antagonize RNA‐processing factors or to recruit various coregulators, thus allowing it to establish a female‐specific gene expression pattern. Here, we summarize the current knowledge about Sxl function and review recent mechanistic and structural studies that further our understanding of how such a seemingly ‘simple’ RNA‐binding protein can exert this plethora of different functions.

Insights into the evolutionary conserved regulation of Rio ATPase activity

Abstract Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure–function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

Special issue: RNA biology in physiology and disease

Genetic information is stored as DNA in nuclear chromatin structures. For protein production, genes are transcribed to mRNA, which transports the information to ribosomes in the cytoplasm. This concept of ‘information flow’ is known for decades but with the advent of new sequencing technologies, it became rapidly apparent that RNA is not only coding but in fact the majority of RNA transcripts within a cell are non-coding. Such RNAs can have diverse functions including scaffolds for large RNA-protein assemblies (RNPs), catalytic activity (rRNA in the ribosome) or the regulation of coding and non-coding gene expression. Any of these non-coding RNAs have specific protein binding partners referred to as RNA-binding proteins (RBPs). For a long time, this extended protein class has not gained much attention. However, with the progress in understanding the function of various non-coding RNAs, these proteins move more and more into our focus. RBPs control the fate of their bound RNAs from synthesis to decay, governing all aspects of RNA biology and dynamically adjusting to cellular requirements. The finding that many RBPs harbor additional functions, e.g. enzymatic activity, has established a close link between RNA biology and in principle unrelated functions such as intermediary metabolism. In this special issue, review articles will address novel findings in RNA biology and discuss the role of RNA metabolism and its regulation in e.g. chronobiology. Furthermore, it will be highlighted in the individual reviews how mis-processing of RNA or mis-regulation of RNA metabolism can change cellular homeostasis tilting the fine balance towards pathology and resulting in disease. Non-coding RNAs such as tRNAs, rRNAs or small nuclear RNAs (snRNAs) are known for a long time and reasonably well understood. The discovery of microRNAs (miRNAs) and other small non-coding RNAs has boosted research into this direction. A plethora of studies has implicated small RNAs in literally all cellular pathways. MiRNAs play important developmental roles in many organisms but are in addition also vital for tissue generation and homeostasis. Furthermore, physiological processes such as insulin secretion and sugar metabolism, fat storage or the function of the cardiovascular system rely on the correct function of specific miRNAs. It is therefore not surprising that miRNAs have been associated with various diseases such as cardiovascular disorders, neurodegenerative disorders or cancer. Review articles within this special issue highlight the function of specific enzymes in miRNA production as well as the diverse sources of small RNAs in human cells. In addition, roles of miRNAs in the nervous system and in smooth muscle homeostasis are summarized. In addition to multiple classes of short regulatory noncoding RNAs, the human genome encodes several thousand long non-protein coding transcripts > 200 nucleotides in length. These long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression in many different cellular pathways and were frequently shown to act as part of protein-containing complexes. They can function as guides and scaffolds for proteins and were shown to act as structural decoys for transcription factors and other cellular molecules. LncRNAs have been implicated in a multitude of different diseases including many types of cancer and are crucial for development and * Jan Medenbach Jan.Medenbach@ur.de