Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex's release in snRNP assembly.

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complex's release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.

Antibodies to extractable nuclear antigens (ENAS) in systemic lupus erythematosus patients: correlations with clinical manifestations and disease activity.

The aim was to explore possible correlations of antibodies to extractable nuclear antigens (ENA) with clinical manifestations and disease activity indices in systemic lupus erythematosus (SLE) patients. A total of 70 consecutive SLE patients (64 females) were included. Disease activity was assessed by SLE activity index (SLEDAI), and British Isles Lupus Assessment Group (BILAG). Anti-Ro/SSA correlated positively with, headache (r=0.24, p=0.04), blurring of vision (r=0.25, p=0.03) and SLEDAI (r=0.25, p=0.04) and negatively with C3 (r=-0.35, p=0.003). Anti-Ro/SSA correlated with anti La/SSB antibodies (r=0.69, p<0.001), but not with anti-DNA, anti-RNP and anti-Sm antibodies. Anti-La/SSB antibodies correlated with headache (r=0.26, p=0.03), SLEDAI (r=0.25, p=0.03) and negatively with C3 (r=-0.34, p=0.004). Anti-La/SSB did not correlate with anti-RNP or anti-Sm antibodies. Anti-Sm antibodies correlated with disease duration (r=0.34, p=0.003), 24 hours urinary proteins (r=0.31, p=0.008), SLEDAI (r=0.31, p=0.009), BILAG renal score (r=0.29, p=0.02) and negatively with age at onset (r=-0.27, p=0.02), WBCs (r=-0.29, p=0.014) and C4 (r=-0.25, p=0.049). In multivariate analyses, anti-Ro/SSA antibodies remained associated with headache, blurring of vision and C3 and anti-La/SSB antibodies remained associated with C3 and with headache. Anti-Sm antibodies were independently associated with disease duration and total SLEDAI scores, while anti-RNP antibodies remained significantly associated with BILAG mucocutaneous scores only. Antibodies to ENAs are associated with clinical aspects of SLE and may play a role in the assessment of disease activity. Insight into these ENAs may lead to new approaches to diagnostic testing, accurate evaluation of disease activity and lead to target approach for SLE.

ART manipulation after controlled ovarian stimulation may not increase the risk of abnormal expression and DNA methylation at some CpG sites of H19,IGF2 and SNRPN in foetuses: a pilot study.

BACKGROUND: To examine the effects of IVF, ICSI and FET, as well as in vitro culture, on the safety of offspring, this study was conducted from the perspective of genetic imprinting to investigate whether assisted reproductive technology would influence the parental and maternal imprinting genes. METHODS: Eighteen foetuses were collected from multifoetal reduction and divided into 6 groups: multifoetal reduction after IVF fresh transferred D3 embryos (n = 3), multifoetal reduction after IVF frozen transferred D3 embryos (n = 3), multifoetal reduction after IVF frozen transferred D5 embryos (n = 3), multifoetal reduction after ICSI fresh transferred D3 embryos (n = 3), multifoetal reduction after ICSI frozen transferred D3 embryos (n = 3), and multifoetal reduction after controlled ovarian hyperstimulation (COH) (n = 3). The imprinted genes H19, IGF2 and SNRPN were selected for analysis. The expression and DNA methylation at some CpG sites of H19, IGF2, and SNRPN were examined using real-time quantitative polymerase chain reaction (PCR) and pyrosequencing. RESULTS: There were no significant differences in the mRNA expression levels among the groups. The mean percentage of H19 methylation (eight CpG sites), IGF2 methylation (five CpG sites) and SNRPN methylation (nine CpG sites) did not differ significantly. CONCLUSIONS: The results suggest that ARTs after controlled ovarian stimulation (IVF, ICSI, cryopreservation and duration of in vitro culture) may not increase the risk of abnormal expression and DNA methylation at some CpG sites of H19, IGF2 and SNRPN in foetuses. Further study with strict design, expanded sample size and CpG sites is essential.

Structural basis for the recognition of spliceosomal SmN/B/B' proteins by the RBM5 OCRE domain in splicing regulation.

The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique ß-sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B'. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B'. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B' proteins in FAS/CD95 alternative splicing regulation.

Methylation profile of SNRPN gene and its correlation with weight and chronological age.

Genomic imprinting is an important epigenetic phenomenon, wherein genes or gene clusters are marked by DNA methylation during gametogenesis. This plays a major role in several functions of normal cells, including cell differentiation, X chromosome inactivation, and the maintenance of chromatin structure, in mammalian development. The aim of this study was to investigate the possible differences in SNRPN gene methylation profiles in non-obese and obese individuals, and in children and adults. Our results did not reveal any statistical correlations between the DNA methylation profiles of the SNRPN gene in children or adult obese and non-obese groups. However, a comparison of the methylation levels with the chronological age revealed statistically significant differences between the means of methylation in adults and children (46.20 ± 5.88 and 39.40 ± 2.87, respectively; P < 0.001). Pearson's correlation analysis indicated a positive association between the level of DNA methylation and the chronological age (R2 = 0.326; P < 0.001). Therefore, we concluded that the methylation profile of the SNRPN promoter (in blood) is not a useful biomarker for determining the predisposition of an individual to obesity. Additionally, we have confirmed that SNRPN methylation increases with age, which raises further questions regarding the role of SNRPN expression during the aging process.

Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

The U1A and U2B″ proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B″ to their respective snRNP is a well-known phenomenon, binding of U2B″ to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B″/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluorescence and isothermal titration calorimetry binding experiments. We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity. In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding. The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

Climbing the vertebrate branch of U1A/U2B″ protein evolution.

In the vertebrate lineage of the U1A/U2B″/SNF protein family, the U1A and U2B″ proteins bind to RNA stem-loops in the U1 or U2 snRNPs, respectively. However, their specialization is fairly recent, as they evolved from a single ancestral protein. The progress of their specialization (subfunctionalization) can be monitored by the amino acid sequence changes that give rise to their modern RNA-binding specificity. Using ancestral sequence reconstruction to predict the intermediates on the evolutionary branch, a probable path of sequential changes is defined for U1A and U2B″. The RNA-binding affinity for U1A/U2B″ protein ancestors was measured using modern U1 and U2 snRNA stem-loops and RNA stem-loop variants to understand how the proteins' RNA specificities evolved.

Resurrection of an Urbilaterian U1A/U2B″/SNF protein.

The U1A/U2B″/SNF family of proteins found in the U1 and U2 spliceosomal small nuclear ribonucleoproteins is highly conserved. In spite of the high degree of sequence and structural conservation, modern members of this protein family have unique RNA binding properties. These differences have necessarily resulted from evolutionary processes, and therefore, we reconstructed the protein phylogeny in order to understand how and when divergence occurred and how protein function has been modulated. Contrary to the conventional understanding of an ancient human U1A/U2B″ gene duplication, we show that the last common ancestor of bilaterians contained a single ancestral protein (URB). The gene for URB was synthesized, the protein was overexpressed and purified, and we assessed RNA binding to modern snRNA sequences. We find that URB binds human and Drosophila U1 snRNA SLII and U2 snRNA SLIV with higher affinity than do modern homologs, suggesting that both Drosophila SNF and human U1A/U2B″ have evolved into weaker binders of one RNA or both RNAs.

Structural basis of assembly chaperone- mediated snRNP formation.

Small nuclear ribonucleoproteins (snRNPs) represent key constituents of major and minor spliceosomes. snRNPs contain a common core, composed of seven Sm proteins bound to snRNA, which forms in a step-wise and factor-mediated reaction. The assembly chaperone pICln initially mediates the formation of an otherwise unstable pentameric Sm protein unit. This so-called 6S complex docks subsequently onto the SMN complex, which removes pICln and enables the transfer of pre-assembled Sm proteins onto snRNA. X-ray crystallography and electron microscopy was used to investigate the structural basis of snRNP assembly. The 6S complex structure identifies pICln as an Sm protein mimic, which enables the topological organization of the Sm pentamer in a closed ring. A second structure of 6S bound to the SMN complex components SMN and Gemin2 uncovers a plausible mechanism of pICln elimination and Sm protein activation for snRNA binding. Our studies reveal how assembly factors facilitate formation of RNA-protein complexes in vivo.

HLA-DR3 restricted T cell epitope mimicry in induction of autoimmune response to lupus-associated antigen SmD.

Although systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder, HLA-D is the most dominant genetic susceptibility locus. This study was undertaken to investigate the hypothesis that microbial peptides bind HLA-DR3 and activate T cells reactive with lupus autoantigens. Using HLA-DR3 transgenic mice and lupus-associated autoantigen SmD protein, SmD(79-93) was identified to contain a dominant HLA-DR3 restricted T cell epitope. This T cell epitope was characterized by using a T-T hybridoma, C1P2, generated from SmD immunized HLA-DR3 transgenic mouse. By pattern search analysis, 20 putative mimicry peptides (P2-P21) of SmD(79-93,) from microbial and human origin were identified. C1P2 cells responded to SmD, SmD(79-93) and a peptide (P20) from Vibro cholerae. Immunization of HLA-DR3 mice with P20 induced T cell responses and IgG antibodies to SmD that were not cross-reactive with the immunogen. A T-T hybridoma, P20P1, generated from P20 immunized mice, not only responded to P20 and SmD(79-93), but also to peptides from Streptococcus agalactiae (P17) and human-La related protein (P11). These three T cell mimics (P20, P11 and P17) induced diverse and different autoantibody response profiles. Our data demonstrates for the first time molecular mimicry at T cell epitope level between lupus-associated autoantigen SmD and microbial peptides. Considering that distinct autoreactive T cell clones were activated by different microbial peptides, molecular mimicry at T cell epitope level can be an important pathway for the activation of autoreactive T cells resulting in the production of autoantibodies. In addition, the novel findings reported herein may have significant implications in the pathogenesis of SLE.

RNA-binding Sm-like proteins of bacteria and archaea. similarity and difference in structure and function.

RNA-binding proteins play a significant role in many processes of RNA metabolism, such as splicing and processing, regulation of DNA transcription and RNA translation, etc. Among the great number of RNA-binding proteins, so-called RNA-chaperones occupy an individual niche; they were named for their ability to assist RNA molecules to gain their accurate native spatial structure. When binding with RNAs, they possess the capability of altering (melting) their secondary structure, thus providing a possibility for formation of necessary intramolecular contacts between individual RNA sites for proper folding. These proteins also have an additional helper function in RNA-RNA and RNA-protein interactions. Members of such class of the RNA-binding protein family are Sm and Sm-like proteins (Sm-Like, LSm). The presence of these proteins in bacteria, archaea, and eukaryotes emphasizes their biological significance. These proteins are now attractive for researchers because of their implication in many processes associated with RNAs in bacterial and archaeal cells. This review is focused on a comparison of architecture of bacterial and archaeal LSm proteins and their interaction with different RNA molecules.

Protein subunit interfaces: A statistical analysis of hot spots in Sm proteins.

The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the properties of Sm protein interfaces with those of a test set, the Binding Interface Database (BID). The comparison revealed that the main differences between the interfaces of Sm proteins and those of the BID set are the content of charged residues, the coordination numbers of the residues, knowledge-based pair potentials, and the conservation scores of hot spots. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surfaces, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Hot spots are residues that make up a small fraction of the interfaces, but they contribute most of the binding energy. These residues are critical to protein-protein interactions. Analyses of knowledge-based pair potentials of hot spot and non-hot spot residues in Sm proteins show that they are significantly different; their mean values are 31.5 and 11.3, respectively. In the BID set, this difference is smaller; in this case, the mean values for hot spot and non-hot spot residues are 20.7 and 12.4, respectively. Hence, the pair potentials of hot spots differ significantly for the Sm and BID data sets. In the interfaces of Sm proteins, the amino acids are tightly packed, and the coordination numbers are larger in Sm proteins than in the BID set for both hot spots and non-hot spots. At the same time, the coordination numbers are higher for hot spots; the average coordination number of the hot spot residues in Sm proteins is 7.7, while it is 6.1 for the non-hot spot residues. The difference in the calculated average conservation score for hot spots and non-hot spots in Sm proteins is significantly larger than it is in the BID set. In Sm proteins, the average conservation score for the hot spots is 7.4. Hot spots are surrounded by residues that are moderately conserved (5.9). The average conservation score for the other interface residues is 5.6. The conservation scores in the BID set do not show a significant distinction between hot and non-hot spots: the mean values for hot and non-hot spot residues are 5.5 and 5.2, respectively. These data show that structurally conserved residues and hot spots are significantly correlated in Sm proteins.

Coilin phosphorylation mediates interaction with SMN and SmB'.

Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.

Interpreting a low resolution map of human U1 snRNP using anomalous scatterers.

We recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 A resolution electron density map. At 5-7 A resolution, alpha helices and beta sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 A. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.

Special Sm core complex functions in assembly of the U2 small nuclear ribonucleoprotein of Trypanosoma brucei.

The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B'' protein from Trypanosoma brucei. U2B'' interacts with a second U2 snRNP protein, U2-40K (U2A'), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.

Structural basis for leucine-rich nuclear export signal recognition by CRM1.

CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 A structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined alpha-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

A survey of nematode SmY RNAs.

SmY RNAs are a family of approximately 70-90 nt small nuclear RNAs found in nematodes. In C. elegans, SmY RNAs copurify in a small ribonucleoprotein (snRNP) complex related to the SL1 and SL2 snRNPs that are involved in nematode mRNA trans-splicing. Here we describe a comprehensive computational analysis of SmY RNA homologs found in the currently available genome sequences. We identify homologs in all sequenced nematode genomes in class Chromadorea. We are unable to identify homologs in a more distantly related nematode species, Trichinella spiralis (class: Dorylaimia), and in representatives of non-nematode phyla that use trans-splicing. Using comparative RNA sequence analysis, we infer a conserved consensus SmY RNA secondary structure consisting of two stems flanking a consensus Sm protein binding site. A representative seed alignment of the SmY RNA family, annotated with the inferred consensus secondary structure, has been deposited with the Rfam RNA families database.

[Identification and expression analysis of a full-length cDNA encoding Brassica napus small nuclear ribonucleoprotein BnSmD1].

By using substractive hybridization (SSH) and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), a full-length cDNA encoding Brassica napus small nuclear ribonucleoprotein, named BnSmD1, was obtained. It had 484 base pairs in length containing an open reading frame (ORF) of 354 bp and encoding a predicted protein of 118 amino acids with a molecular weight of 13 kDa. The BnSmD1 protein shares two highly conserved Sm folds (Sm-1 and Sm-2) and a C-terminal RG dipeptide repeat. Northern blot analysis revealed that BnSmD1 was expressed in all tested organs in B. napus, but its transcript level in early floral buds was much higher than that in leaf and stem tissues. No obvious expression difference was observed in leaf and stem tissues between the apetalous line Apet33-10 petalled near-isogenic line Pet33-10. Compared with wild type, the expression of BnSmD1 in the early floral buds of apetalous mutant Apet33-10 was significantly reduced. Taken together, our results suggest that BnSmD1 may play an important role in early floral petal development in B. napus.