A syn-anti conformational difference allows SRSF2 to recognize guanines and cytosines equally well.

SRSF2 (SC35) is a key player in the regulation of alternative splicing events and binds degenerated RNA sequences with similar affinity in nanomolar range. We have determined the solution structure of the SRSF2 RRM bound to the 5'-UCCAGU-3' and 5'-UGGAGU-3' RNA, both identified as SRSF2 binding sites in the HIV-1 tat exon 2. RNA recognition is achieved through a novel sandwich-like structure with both termini forming a positively charged cavity to accommodate the four central nucleotides. To bind both RNA sequences equally well, SRSF2 forms a nearly identical network of intermolecular interactions by simply flipping the bases of the two consecutive C or G nucleotides into either anti or syn conformation. We validate this unusual mode of RNA recognition functionally by in-vitro and in-vivo splicing assays and propose a 5'-SSNG-3' (S=C/G) high-affinity binding consensus sequence for SRSF2. In conclusion, in addition to describe for the first time the RNA recognition mode of SRSF2, we provide the precise consensus sequence to identify new putative binding sites for this splicing factor.

Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1.

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5'end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5'end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.

Isolated pseudo-RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition.

Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the ß-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression.

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5' and 3' ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5' and the 3' pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.

Functional kinomics establishes a critical node of volume-sensitive cation-Cl- cotransporter regulation in the mammalian brain.

Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation - a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl--sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl- uptake and stimulation of KCC3-mediated Cl- extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought "Cl-/volume-sensitive kinase" of the cation-Cl- cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.

SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition.

Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis.

RRM-RNA recognition: NMR or crystallography and new findings.

To characterize protein-RNA recognition at the molecular level, structural biology has turned out to be an indispensable approach. Detailed and direct insights into the mechanism of RNA binding and specificity have emerged from protein-RNA structures, especially from the most abundant RNA recognition motif (RRM). Although this protein domain has a very conserved α-ß fold, it can recognize a large number of different RNA sequences and shapes and can be involved in a multitude of biological processes. Here, we report on recent single and multiple RRM-RNA structures and point out those features that provide novel insights into the mechanism of RNA recognition by RRMs. We further outline inherent problems to both NMR spectroscopy and X-ray crystallography methods and review recent strategies that emphasize the need to use both methodologies for more rapid and accurate structure determinations.