The sequence-specific RNA-binding proteins SRp20 and 9G8 are the smallest members of the serine-and arginine-rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5 0 CAUC3 0 . The SRp20-RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5 0 cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine-rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip-associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.
The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded b-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix a1, together with amino acids from strand b2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.
The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N-and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.
SignificanceNanotechnology-based imaging is expected to bring breakthroughs in cancer diagnosis by improving imaging sensitivity and specificity while reducing toxicity. Here, we developed an innovative nanosystem for positron emission tomography (PET) imaging based on a self-assembling amphiphilic dendrimer. This dendrimer assembled spontaneously into uniform supramolecular nanomicelles with abundant PET reporting units on the surface. By harnessing both dendrimeric multivalence and the “enhanced permeation and retention” (EPR) effect, this dendrimer nanosystem effectively accumulated in tumors, leading to exceedingly sensitive and specific imaging of various tumors, especially those that are otherwise undetectable using the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). This study illustrates the power of nanotechnology based on self-assembling dendrimers to provide an effective platform for bioimaging and related biomedical applications.
Cytological analysis of thyroid nodules detected using ultrasound-guided fine-needle aspiration technique is an efficient method for the diagnosis of well-differenciated tumors such as papillary thyroid carcinoma. However, for between 10 to 30% of all the nodules, the cytological analysis based on fine-needle aspiration biopsies leads to an "indeterminated" identification. Consequently, a surgical excision is then necessary for a definite histological diagnosis of the lesions, resulting in 85% of the patient with indeterminated nodules undergoing unnecessary surgery since their tumor is finally diagnosed as benign. In this work, we discuss how HRMAS (1)H NMR-based metabolomics could be a complementary tool for the diagnosis of these elusive cases. We first showed that our approach was able to discriminate clearly any types of thyroid lesions from healthy tissues. Then we proceeded to demonstrate that the information produced by (1)H HRMAS NMR spectra differentiate tumors according to their malignancy grade, even when they belong to the "indeterminate" category. Analysis of the discriminating spectral area in this last case points out toward a possible increase of phenylalanine, taurine, and lactate and a decrease of choline and choline derivatives, myo- and scyllo-inositol in the malignant tumors compared to the benign ones.
Functional materials capable of responding to stimuli intrinsic to diseases are extremely important for specific drug delivery at the disease site. However, developing on-demand stimulus-responsive vectors for targeted delivery is highly challenging. Here, we report a stimulus-responsive fluorinated bola-amphiphilic dendrimer for on-demand delivery of small interfering RNA (siRNA) in response to the characteristic high level of reactive oxygen species (ROS) in cancer cells. This dendrimer bears a ROS-sensitive thioacetal in the hydrophobic core and positively charged poly(amidoamine) dendrons at the terminals, capable of interacting and compacting the negatively charged siRNA into nanoparticles to protect the siRNA and promote cellular uptake. The ROS-sensitive feature of this dendrimer boosted specific and efficient disassembly of the siRNA/vector complexes in ROS-rich cancer cells for effective siRNA delivery and gene silencing. Moreover, the fluorine tags in the vector enabled 19 F-NMR analysis of the ROS-responsive delivery process. In addition, this ingenious and distinct bolaamphiphilic dendrimer is also able to combine the advantageous delivery features of both lipid and dendrimer vectors. Therefore, it represents an innovative on-demand stimulus-responsive delivery platform based on dendrimer molecular engineering.
Conspectus Dendrimers, notable for their well-defined radial structures with numerous terminal functionalities, hold great promise for biomedical applications such as drug delivery, diagnostics, and therapeutics. However, their translation into clinical use has been greatly impeded by their challenging stepwise synthesis and difficult purification. To circumvent these obstacles, we have pioneered a self-assembly approach to constructing noncovalent supramolecular dendrimers using small amphiphilic dendrimer building units which can be easily synthesized and purified. By virtue of their amphipathic nature, the small amphiphilic dendrimers are able to self-assemble and generate large supramolecular dendrimers via noncovalent weak interactions such as van der Waals forces, H bonds, and electrostatic interactions. The so-created noncovalent dendrimers can mimic covalent dendrimers not only in terms of the radial structural feature emanating from a central core but also in their capacity to deliver drugs and imaging agents for biomedical applications. The noncovalent supramolecular dendrimers can be easily synthesized and modulated with regard to size, shape, and properties by varying the nature of the hydrophobic and hydrophilic entities as well as the dendrimer generation and terminal functionalities, ensuring their adaptability to specific applications. In particular, the dendritic structure of the amphiphilic building units permits the creation of large void spaces within the formed supramolecular dendrimers for the physical encapsulation of drugs, while the large number of surface functionalities can be exploited for both physical and chemical conjugation of pharmaceutic agents for drug delivery. Poly(amidoamine) (PAMAM) dendrimers are the most intensively studied for biomedical applications by virtue of their excellent biocompatibility imparted by their peptide-mimicking amide backbones and numerous interior and terminal amine functionalities. We present a short overview of our self-assembly strategy for constructing supramolecular PAMAM dendrimers for biomedical applications. Specifically, we start with the introduction of dendrimers and their synthesis, focusing on the innovative self-assembly synthesis of supramolecular dendrimers. We then detail the representative examples of the noncovalent supramolecular PAMAM dendrimers established in our group for the delivery of anticancer drugs, nucleic acid therapeutics, and imaging agents, either within the dendrimer interior or at the dendrimer terminals on the surface. Some of the supramolecular dendrimer nanosystems exhibit outstanding performance, excelling the corresponding clinical anticancer therapeutics and imaging agents. This self-assembly approach to creating supramolecular dendrimers is completely novel in concept yet easy to implement in practice, offering a fresh perspective for exploiting the advantageous features of dendrimers in biomedical applications.
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