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).

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 ...