Prevalent RNA recognition motif duplication in the human genome

The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.     

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.     

RRM RNA结合蛋白的结构与功能

RRM RNA结合蛋白是一类含一个或数个RRM结构域及附属结构域的RNA结合蛋白,参与RNA前体的剪接、RNA的细胞定位、RNA的稳定性等多种转录后调控过程.在RRM基序中含有许多保守的氨基酸以保证对RNA的结合活性,但是这一家族的不同蛋白质却能特异地结合各种不同的RNA分子.RRM RNA结合蛋白与某些人类遗传性疾病及肿瘤相关.     

Solution structure of the second RNA recognition motif (RRM) domain of murine T cell intracellular antigen-1 (TIA-1) and its RNA recognition mode

T cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (betaalphabetabetaalphabeta) and possesses an extra beta-strand between beta2 and beta3, which forms an additional beta-sheet with the C-terminal part of beta2. We refer to this structure as the beta2-beta2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta2-beta2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta2-beta2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.     

Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization

RNA binding proteins (RBPs) play an important role in mRNA metabolism including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements, or zipcodes. Putative trans-acting RBPs that recognize prolamine RNA zipcodes required for restricted localization to protein body-ER have previously been identified. Here, we describe the identification of RBP-P using a Northwestern blot approach as an RBP that recognizes and binds to glutelin zipcode RNA, which is required for proper RNA localization to cisternal-ER. RBP-P protein expression coincides with that of glutelin during seed maturation and is localized to both the nucleus and cytosol. RNA-immunoprecipitation and subsequent RT-PCR analysis further demonstrated that RBP-P interacts with glutelin RNAs. In vitro RNA–protein UV-crosslinking assays showed that recombinant RBP-P binds strongly to glutelin mRNA, and in particular, 3′ UTR and zipcode RNA. RBP-P also exhibited strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in mRNA splicing. Overall, these results support a multifunctional role for RBP-P in glutelin mRNA metabolism, perhaps in nuclear pre-mRNA splicing and cytosolic localization to the cisternal-ER.     

小RNA与蛋白质的相互作用

小分子调控RNA,包括siRNA（small interfering RNA）、miRNA（microRNA）和piRNA（piwiinteracting RNA）、hsRNA（heterochromatin associatedsmall RNA）等,是当前生命科学研究的前沿热点。越来越多的证据表明,这些小分子RNA存在于几乎所有较高等的真核生物细胞中,对生物体具有非常重要的调控功能。它们通过各种序列特异性的RNA基因沉默作用,包括RNA干扰（RNAi）、翻译抑制、异染色质形成等,调控诸如生长发育、应激反应、沉默转座子等各种各样的细胞进程。随着对这些小分子调控RNA的发现,一些RNascⅢ酶家族成员、Argonaute蛋白质家族成员及RNA结合蛋白质等先后被鉴定为小RNA的胞内蛋白质合作者,参与小RNA的加工成熟和在细胞内行使功能。本综述简介一些RNA沉默作用途径中重要组分的结构和功能的研究进展。     

Two different RNA binding activities for the AU-rich element and the poly(A) sequence of the mouse neuronal protein mHuC.

HuC is one of the RNA binding proteins which are suggested to play important roles in neuronal differentiation and maintenance. We cloned and sequenced cDNAs encoding a mouse protein which is homologous to human HuC (hHuC). The longest cDNA encodes a 367 amino acid protein with three RNA recognition motifs (RRMs) and displays 96% identity to hHuC. Northern blot analysis showed that two different mRNAs, of 5.3 and 4.3 kb, for mouse HuC (mHuC) are expressed specifically in brain tissue. Comparison of cDNA sequences with the corresponding genomic sequence revealed that alternative 3' splice site selection generates two closely related mHuC isoforms. Iterative in vitro RNA selection and binding analyses showed that both HuC isoforms can bind with almost identical specificity to sequences similar to the AU-rich element (ARE), which is involved in the regulation of mRNA stability. Functional domain mapping using mHuC deletion mutants showed that the first RRM binds to ARE, that the second RRM has no RNA binding activity by itself, but facilitates ARE binding by the first RRM and that the third RRM has specific binding activity for the poly(A) sequence.     

The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding

Prp24 is an essential yeast U6 snRNP protein with four RNA recognition motifs (RRMs) that facilitates the association of U4 and U6 snRNPs during spliceosome assembly. Genetic interactions led to the proposal that RRMs 2 and 3 of Prp24 bind U6 RNA, while RRMs 1 and 4 bind U4 RNA. However, the function of each RRM has yet to be established through biochemical means. We compared the binding of recombinant full-length Prp24 and truncated forms lacking RRM 1 or RRM 4 with U6 RNA. Contrary to expectations, we found that the N-terminal segment containing RRM 1 is important for high-affinity binding to U6 RNA and for discrimination between wild-type U6 RNA and U6 with point mutations in the 3' intramolecular stem-loop. In contrast, deletion of RRM 4 and the C terminus did not significantly alter the affinity for U6 RNA, but resulted in the formation of higher order Prp24.U6 complexes. Truncation and internal deletion of U6 RNA mapped three Prp24-binding sites, with the central site providing most of the affinity for Prp24. A newly identified temperature-sensitive lethal point mutation in RRM 1 is exacerbated by mutations in the U6 RNA telestem, as is a mutation in RRM 2, but not one in RRM 3. We propose that RRMs 1 and 2 of yeast Prp24 bind the same central site in U6 RNA that is bound by the two RRMs of human Prp24, and that RRMs 3 and 4 bind lower affinity flanking sites, thereby restricting the stoichiometry of Prp24 binding.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.