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). We have developed the Phenol Toluol extraction (PTex) protocol 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 global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis.

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.

RNA-binding proteins (RBPs) are key regulators in post-transcriptional control of gene expression. Mutations that alter their activity or abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression. Here we review RBP-mediated autogenous feedback regulation that either serves to maintain protein abundance within a physiological range (by negative feedback) or generates binary, genetic on/off switches important for e.g. cell fate decisions (by positive feedback).

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.small nuclear RNA ͉ small nuclear ribonucleoprotein ͉ splicing ͉ genetic screen ͉ thymus M essenger RNA splicing requires the ordered assembly of the spliceosome from Ͼ100 protein components and five small nuclear RNAs (snRNAs): U1, U2, U4, U5, and U6 (reviewed in refs. 1-3). After splicing catalysis and mRNA release, the spliceosome disassembles, and its components undergo a recycling phase, which still is poorly understood. In humans, recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) to functional U4/U6 snRNPs requires in vitro p110/SART3, a general splicing factor referred to as p110 in the present article (4, 5). In addition, p110 functions in recycling of the U4atac/U6atac snRNP (6). Characteristically, p110 associates only transiently with the U6 and U4/U6 snRNPs but is absent from the U4/U6.U5 tri-snRNP and spliceosomes.The domain structure of the human p110 protein is composed of at least seven tetratricopeptide repeats (TPR) in the Nterminal half, followed by two RNA recognition motifs (RRMs) in the C-terminal half, as well as a stretch of 10 highly conserved amino acids at the C terminus (C10 domain). The N-terminal TPR domain functions in interaction with the U4/U6 snRNPspecific 90K protein, the RRMs are important for U6 snRNA binding, and the conserved C10 domain is critical for interacting with the U6-specific LSm proteins (5,7,8). Thus, multiple contacts mediate the interaction between p110 and the U4 and U6 components.This p110 domain organization is conserved in many other eukaryotes, including Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and Drosophila melanogaster (5). The Saccharomyces cerevisiae Prp24 protein, although functionally related to human p110, is an exception in that it lacks the entire N-terminal half with the TPR domain (9).Here we use the zebrafish system to study the system-wide role and in vivo function of p110. We describe the phenotype of a zebrafish mutant, called earl grey (egy), that originated from a genetic screen for mutants of T cell and thymus development. Surprisingly, the embryonically lethal mutation was mapp...

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 proteinprotein interactions.Nuclear pre-mRNA splicing takes places in a large RNP complex, the spliceosome, which is assembled in an ordered multistep process. It consists of five small nuclear RNAs (the U1, U2, U4, U5, and U6 snRNAs) and more than 100 proteins, as recent proteomic analyses have determined (15,16,39). The spliceosome shows characteristic dynamics during assembly and splicing catalysis. For example, only the U2, U5, and U6 snRNAs participate in the catalytic center of the spliceosome, whereas the U1 and U4 snRNAs play essential roles only during the early assembly stages. After completion of the twostep splicing reaction and the release of mRNA and lariat products, the spliceosome disassembles into its components. Before entering a new cycle, at least some the components presumably must be reactivated. However, very little is known about this recycling phase of the spliceosome cycle.The U4, U5, and U6 snRNAs enter the prespliceosome in the form of the 25S U4/U6.U5 tri-snRNP but are released from the spliceosome in their singular forms, the U4 and U6 snRNPs. These interact with each other to regenerate the U4/U6 di-snRNP, in which the two snRNAs are stably base paired (see Fig. 6C). The addition of the U5 snRNP generates the U4/U6.U5 tri-snRNP, which is integrated into the spliceosome. During each spliceosome cycle, the participating snRNAs undergo extensive structural rearrangements governed by specific protein factors (for reviews, see references 7, 27, 28, and 33).Regarding the molecular organization of the mammalian U4/U6 snRNP, a hierarchical assembly pathway has been demonstrated; this pathway also is conserved in the related U4atac/ U6atac snRNP of the minor spliceosome (24). Of the five specific protein components, the 15.5K protein initially recognizes the loop region of the U4 5Ј stem-loop (23), followed by binding of the 61K protein. The subsequent integration of the ...

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.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR‐mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one‐carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR‐driven changes to gene expression and resistance to pharmacological treatment.