Sm Protein Binding Research Articles

Structure of the Sm Binding Site From Human U4 snRNA Derived From a 3 ns PME Molecular Dynamics Simulation

A molecular dynamics simulation of the Sm binding site from human U4 snRNA was undertaken to determine the conformational flexibility of this region and to identify RNA conformations that were important for binding of the Sm proteins. The RNA was fully-solvated (>9,000 water molecules) and charge neutralized by inclusion of potassium ions. A three nanosecond MD simulation was conducted using AMBER with long-range electrostatic forces considered using the particle mesh Ewald summation method. The initial model of the Sm binding site region had the central and 3′ stem-loops that flanked the Sm site co-axial with one another, and with the single-stranded Sm binding site region ([I] conformation). During the course of the trajectory, the axes of the 3′ stem-loop, and later the central stem-loop, became roughly orthogonal from their original anti-parallel orientation. As these conformational changes occurred, the snRNA adopted first an [L] conformation, and finally a [U] conformation. The [U] conformation was more stable than either the [I] or [L] conformations, and persisted for the final 1 ns of the trajectory. Analysis of the structure resulting from the MD simulations revealed the bulged nucleotide, U114, and the mismatched A91-G110 base pair provided distinctive structural features that may enhance Sm protein binding. Based on the results of the MD simulation and the available experimental data, we proposed a mechanism for the binding of the Sm protein sub-complexes to the snRNA. In this model, the D1/D2 and E/F/G Sm protein sub-complexes first bind the snRNA in the [U] conformation, followed by conformational re-arrangement to the [I] conformation and binding of the D3/B Sm protein sub-complex.

U snRNP assembly in yeast involves the La protein.

In all eukaryotic nuclei, the La autoantigen binds nascent RNA polymerase III transcripts, stabilizing these RNAs against exonucleases. Here we report that the La protein also functions in the assembly of certain RNA polymerase II-transcribed RNAs into RNPs. A mutation in a core protein of the spliceosomal snRNPs, Smd1p, causes yeast cells to require the La protein Lhp1p for growth at low temperatures. Precursors to U1, U2, U4 and U5 RNAs are bound by Lhp1p in both wild-type and mutant cells. At the permissive temperature, smd1-1 cells contain higher levels of stable U1 and U5 snRNPs when Lhp1p is present. At low temperatures, Lhp1p becomes essential for the accumulation of U4/U6 snRNPs and for cell viability. When U4 RNA is added to extracts, the pre-U4 RNA, but not the mature RNA, is bound by Smd1p. These results suggest that, by stabilizing a 3'-extended form of U4 RNA, Lhp1p facilitates efficient Sm protein binding, thus assisting formation of the U4/U6 snRNP.

Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.

Intragenic telSMN Mutations: Frequency, Distribution, Evidence of a Founder Effect, and Modification of the Spinal Muscular Atrophy Phenotype by cenSMN Copy Number

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.

Inhibition of fibrillin 1 expression using U1 snRNA as a vehicle for the presentation of antisense targeting sequence.

This study examines whether the mimicking of selected properties of naturally occurring antisense RNAs in prokaryotes allows efficient inhibition of gene expression by in situ-expressed recombinant molecules in mammalian cells. Prokaryotic regulatory transcripts are expressed at high levels and have hairpin structures at their termini, features reminiscent of small nuclear RNAs (snRNAs) which are abundant and stable in the nucleus of all mammalian cells. A sequence complementary to fibrillin-1 (FBN1) mRNA, interrupted in its center by a hammerhead ribozyme, was substituted for the Sm protein binding site between the stem-loop structures of U1 snRNA. Expression of the chimeric antisense RNA resulted in dramatic inhibition of expression of fibrillin-1 message and protein in stably transfected cultured cells. The inhibitory effect was localized to the nucleus. The biological properties of U1 snRNA may provide a widely applicable vehicle for the in vivo delivery of antisense targeting sequences.

Domain Analysis of Human U5 RNA CAP TRIMETHYLATION, PROTEIN BINDING, AND SPLICEOSOME ASSEMBLY

We have analyzed the sequence requirements of the human U5 RNA during small nuclear ribonucleoprotein (snRNP) and spliceosome assembly. A collection of mutant derivatives of the human U5 RNA gene was constructed in a U1 expression vector and transiently transfected in mammalian cells. Using immunoprecipitation and affinity selection assays, the cap trimethylation, the binding of Sm proteins and of the U5 snRNP-specific protein p220, as well as the assembly of the U4/U5/U6 triple snRNP and of spliceosomes were determined. By mutational analysis we were able to assign distinct functions to several structural elements of the human U5 RNA. Efficient binding of the Sm proteins requires the 3' stem-loop. Both the Sm protein-binding site and the 3' stem-loop are necessary for the formation of the trimethyl guanosine cap, consistent with Sm protein binding being a prerequisite for cap trimethylation. Specific elements of the U5 RNA 5' stem-loop contribute to efficient p220 association, in particular stem Ib. Interestingly, the highly conserved loop I appears to be a multifunctional element; in addition to its function in splice-site selection the 5' loop is involved in binding of p220 and in the assembly of the U4/U5/U6 triple snRNP. In sum, this mutational analysis has identified four functional domains of the human U5 RNA.

Architecture of the U5 Small Nuclear RNA

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Architecture of the U5 small nuclear RNA.

We have used comparative sequence analysis and deletion analysis to examine the secondary structure of the U5 small nuclear RNA (snRNA), an essential component of the pre-mRNA splicing apparatus. The secondary structure of Saccharomyces cerevisiae U5 snRNA was studied in detail, while sequences from six other fungal species were included in the phylogenetic analysis. Our results indicate that fungal U5 snRNAs, like their counterparts from other taxa, can be folded into a secondary structure characterized by a highly conserved stem-loop (stem-loop 1) that is flanked by a moderately conserved internal loop (internal loop 1). In addition, several of the fungal U5 snRNAs include a novel stem-loop structure (ca. 30 nucleotides) that is adjacent to stem-loop 1. By deletion analysis of the S. cerevisiae snRNA, we have demonstrated that the minimal U5 snRNA that can complement the lethal phenotype of a U5 gene disruption consists of (i) stem-loop 1, (ii) internal loop 1, (iii) a stem-closing internal loop 1, and (iv) the conserved Sm protein binding site. Remarkably, all essential, U5-specific primary sequence elements are encoded by a 39-nucleotide domain consisting of stem-loop 1 and internal loop 1. This domain must, therefore, contain all U5-specific sequences that are essential for splicing activity, including binding sites for U5-specific proteins.

Pseudourine formation in small nuclear RNAs

Recent in vitro studies on the formation of pseudouridine (Ψ) in the spliceosomal small nuclear RNAs (snRNA) are reviewed. Multiple Ψ synthase activities, in some cases more that one per snRNA, are responsible for this modification of uridine. There is a requirement for Sm protein binding for the efficient formation of Ψ in U5 RNA but not for the modification of U2 RNA. The inhibition of Ψ formation by the incorporation of 5-fluorouridine in the snRNA is also reviewed.

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.

Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site.

Human autoantibodies of the Sm specificity recognize a conserved set of proteins found in the U class small nuclear ribonucleoproteins (U snRNPs), key trans-acting factors involved in the splicing of mRNA precursors. The Sm protein binding site in U snRNAs is unusual because of its single-stranded nature and its simple sequence motif (AU5-6GPu). Here we use genetics to probe this specific protein-RNA interaction by saturation mutagenesis of the Sm binding site of the Saccharomyces cerevisiae U5 snRNA. The assay system used to analyze these mutations takes advantage of a conditionally expressed U5 gene which does not support growth under non-permissive conditions; U5 genes containing Sm site mutations were tested for their ability to complement this lethal phenotype. Our results indicate that the Sm binding site is remarkably tolerant to mutation despite its high degree of conservation, suggesting that relatively few or redundant specific contacts can determine recognition of single-stranded RNA by protein. A complementary biochemical analysis of these mutants demonstrates that integrity of the Sm site is necessary for snRNP stability in vivo and in vitro.

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

Regulated 3? processing of histone mRNA requires both the U7 snRNP and a heat-labile component with interesting properties

U7-snRNA is an essential component of the RNA processing machinery which generates the 3′ end of mature histone mRNA in the sea urchin. U7-snRNP is classified as a member of the Sm-type U-snRNP family by virtue of its recognition by both anti-thrimethylguanosine and anti-Sm antibodies. We have analyzed the function/structure relationship of the U7-snRNP by mutagenesis experiments. These suggest that the U7-snRNP particle of the sea urchin has three important domains. 1. The 5′ terminal sequences, nucleotides 1–9, are accessible to micronuclease while the remainder of the RNA is highly protected and hence presumably bound up with proteins. The complementarities of the U7-RNA 5′ terminal sequences to the histone pre-mRNA are of functional importance. 2. Nucleotides 9–20 contain a sequence mediating Sm-protein binding. The complementarities between the U7-RNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a minor, if any, role in 3′ processing. 3. The terminal palindrome of U7-RNA must be maintained as structure in order for the U7-RNP to work, but its sequence can be drastically altered without any observable effect on snRNP assembly or in 3′ processing.