U1 snRNPs Research Articles

Biogenesis and Functions of Circular RNAs Come into Focus.

Many eukaryotic protein-coding genes are able to generate exonic circular RNAs. Most of these covalently linked transcripts are expressed at low levels, but some accumulate to higher levels than their associated linear mRNAs. We highlight several methodologies that have been developed in recent years to identify and characterize these transcripts, and which have revealed an increasingly detailed view of how circular RNAs can be generated and function. It is now clear that modulation of circular RNA levels can result in a variety of molecular and physiological phenotypes, including effects on the nervous system, innate immunity, microRNAs, and many disease-relevant pathways.

Identification of a noncanonical RNA binding domain in the U2 snRNP protein SF3A1.

During splicing of pre-mRNA, 5′ and 3′ splice sites are brought within proximity by interactions between the pre-mRNA bound U1 and U2 snRNPs, followed by recruitment of the tri-snRNP for assembly of the mature spliceosome. Previously, we identified an interaction between the U2 snRNP-specific protein SF3A1 and the stem–loop 4 (SL4) of the U1 snRNA that occurs during the early steps of spliceosome assembly. Although harboring many annotated domains, SF3A1 lacks a canonical RNA binding domain. To identify the U1-SL4 binding region in SF3A1, we expressed amino- and carboxy-terminal deletion constructs using a HeLa cell-based cell free expression system. UV-crosslinking of the truncated proteins with 32P-U1-SL4 RNA identified the carboxy-terminal ubiquitin-like (UBL) domain of SF3A1 as the RNA binding region. Characterization of the interaction between SF3A1-UBL and U1-SL4 by electrophoretic mobility shift assay and surface plasmon resonance determined high binding affinity (KD = ∼97 nM), and revealed the double-stranded G-C rich stem of U1-SL4 as an important feature for binding to the UBL domain. Further, mutations of two conserved tyrosine residues, Y772 and Y773, were found to cause a two- and fivefold decrease in the binding affinity for U1-SL4, respectively. Finally, we found that SF3A1-UBL can specifically pull down the U1 snRNP from HeLa nuclear extract, demonstrating its capacity to bind U1-SL4 in the context of the intact snRNP. Thus, the data show that the UBL domain of SF3A1 can function as an RNA binding domain and that mutations in this region may interfere with U1-SL4 binding.

Spt5 modulates cotranscriptional spliceosome assembly in Saccharomyces cerevisiae.

There is increasing evidence from yeast to humans that pre-mRNA splicing occurs mainly cotranscriptionally, such that splicing and transcription are functionally coupled. Currently, there is little insight into the contribution of the core transcription elongation machinery to cotranscriptional spliceosome assembly and pre-mRNA splicing. Spt5 is a member of the core transcription elongation machinery and an essential protein, whose absence in budding yeast causes defects in pre-mRNA splicing. To determine how Spt5 affects pre-mRNA splicing, we used the auxin-inducible degron system to conditionally deplete Spt5 in Saccharomyces cerevisiae and assayed effects on cotranscriptional spliceosome assembly and splicing. We show that Spt5 is needed for efficient splicing and for the accumulation of U5 snRNPs at intron-containing genes, and therefore for stable cotranscriptional assembly of spliceosomes. The defect in cotranscriptional spliceosome assembly can explain the relatively mild splicing defect as being a consequence of the failure of cotranscriptional splicing. Coimmunoprecipitation of Spt5 with core spliceosomal proteins and all spliceosomal snRNAs suggests a model whereby Spt5 promotes cotranscriptional pre-mRNA splicing by stabilizing the association of U5 snRNP with spliceosome complexes as they assemble on the nascent transcript. If this phenomenon is conserved in higher eukaryotes, it has the potential to be important for cotranscriptional regulation of alternative splicing.

Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A

Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.

Structural insight into pre‐mRNA splicing catalyzed by the spliceosome

Pre‐mRNA splicing is catalyzed by the spliceosome, a huge protein‐RNA complex composed of the U1, U2, U4, U5, U6 snRNPs and many non‐snRNP related proteins. U1 snRNP plays a critical role in 5′ splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. We have recently determined the structure of yeast U1 snRNP at 3.6Å resolution using cryoEM. The structure reveals common features as well as important differences from the human U1 snRNP. It provides atomic models of nearly all essential domains of U1 snRNA, all core proteins, and five auxiliary proteins. The structure offers a framework to integrate a wealth of existing genetic and biochemical data regarding the structure and function of yeast U1 snRNP and the mechanism of 5′ ss recognition. In addition, the yeast U1 snRNP structure and biochemical analyses based on the structure provided intriguing insight into the structure and function of these auxiliary human U1 snRNP proteins in alternative splicing in higher eukaryotes.The spliceosome undergoes dramatic changes signified by the E, A, Pre‐B, B, Bact, B*, C, C*, P, and ILS complexes in a splicing cycle. CryoEM structures of B, Bact, C, C*, and ILS revealed mechanisms of 5′ ss recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release, all related to the post‐catalytic P complex. We have recently determined the cryoEM structure of the yeast P complex at 3.3Å resolution, revealing that the 3′ ss recognition is driven by the interaction between 3′ and 5′ ss, likely facilitated by a stem‐like structure formed in the intronic regions between the branch site and 3′ ss. The structure shows that a new protein(s) becomes stably associated with the core components of the P complex around Prp8 and Prp22, securing the 3′ exon and potentially regulating the activity of Prp22. The structure demonstrates that Prp22 binds 15–21 nucleotides downstream of the exon‐exon junction, enabling it to pull the 3′ intron‐exon and ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading and exon release, respectively. By providing insights into the 3′ ss recognition, exon ligation, and the action of RNA helicase Prp22, the P complex structure fills a major gap in our understanding of the splicing cycle.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Architecture of the U6 snRNP reveals specific recognition of 3′-end processed U6 snRNA

The spliceosome removes introns from precursor messenger RNA (pre-mRNA) to produce mature mRNA. Prior to catalysis, spliceosomes are assembled de novo onto pre-mRNA substrates. During this assembly process, U6 small nuclear RNA (snRNA) undergoes extensive structural remodeling. The early stages of this remodeling process are chaperoned by U6 snRNP proteins Prp24 and the Lsm2–8 heteroheptameric ring. We now report a structure of the U6 snRNP from Saccharomyces cerevisiae. The structure reveals protein–protein contacts that position Lsm2–8 in close proximity to the chaperone “active site” of Prp24. The structure also shows how the Lsm2–8 ring specifically recognizes U6 snRNA that has been post-transcriptionally modified at its 3′ end, thereby elucidating the mechanism by which U6 snRNPs selectively recruit 3′ end-processed U6 snRNA into spliceosomes. Additionally, the structure reveals unanticipated homology between the C-terminal regions of Lsm8 and the cytoplasmic Lsm1 protein involved in mRNA decay. The spliceosome removes introns from precursor messenger RNAs to produce mature mRNAs. Here the authors report a U6 snRNP crystal structure that provides insight on how the 3′ phosphate of U6 snRNA is recognized by the Lsm2–8 complex and how the U6 snRNP proteins sequester and protect the active site RNA until it is assembled into the spliceosome.

Prespliceosome structure provides insights into spliceosome assembly and regulation

The spliceosome catalyzes the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors1. For branching, the intron 5'-splice site (5'SS) and the branch point (BP) sequence are selected and brought into the prespliceosome by the U1 and U2 snRNPs1, which is a focal point for the regulation by alternative splicing factors2. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions3. However, the structural basis of early spliceosome assembly events remains poorly understood4. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5'SS in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8) that are linked to human disease5,6. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5'SS transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B complex spliceosome7,8. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.

Structural basis for spliceosome assembly

Molecular Biology The spliceosome removes noncoding sequences from precursor RNA and ligates coding sequences into useful mRNA. The pre-spliceosome (A complex) associates with a small nuclear ribonucleoprotein (snRNP) complex called U4/U6.U5 tri-snRNP to form the pre-B complex, which is converted into the precatalytic B complex. Bai et al. solved the cryo–electron microscopy structures of the pre-B and B complexes isolated from yeast. These structures show the U1 and U2 snRNPs and allow modeling of the A complex to reveal the early steps of spliceosome assembly and activation. Science , this issue p. [1423][1] [1]: /lookup/doi/10.1126/science.aau0325

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation.

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo-electron microscopy structures of the Saccharomyces cerevisiae pre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5' splice site (5'SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5'-exon-5'SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

Architecture of the U6 snRNP reveals specific recognition of 3\u2032-end processed U6 snRNA

The spliceosome removes introns from precursor messenger RNA (pre-mRNA) to produce mature mRNA. Prior to catalysis, spliceosomes are assembled de novo onto pre-mRNA substrates. During this assembly process, U6 small nuclear RNA (snRNA) undergoes extensive structural remodeling. The early stages of this remodeling process are chaperoned by U6 snRNP proteins Prp24 and the Lsm2–8 heteroheptameric ring. We now report a structure of the U6 snRNP from Saccharomyces cerevisiae. The structure reveals protein–protein contacts that position Lsm2–8 in close proximity to the chaperone “active site” of Prp24. The structure also shows how the Lsm2–8 ring specifically recognizes U6 snRNA that has been post-transcriptionally modified at its 3′ end, thereby elucidating the mechanism by which U6 snRNPs selectively recruit 3′ end-processed U6 snRNA into spliceosomes. Additionally, the structure reveals unanticipated homology between the C-terminal regions of Lsm8 and the cytoplasmic Lsm1 protein involved in mRNA decay.

The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein Expression

The circadian factor BMAL1 is a fundamental transcriptional regulator of cell time keeping and expression of cell specific genes important for homeostasis. Our lab developed the inducible, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal muscle. Skeletal muscles from these mice exhibit decreased specific tension and reduced unstimulated tension developed during a fatigability test. Since this change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in iMSBmal1+/+). RNA-Seq from these muscles indicates increased inclusion of titin exons 150-155 and 190-225, which are found within titin's PEVK region. Because this region is highly extensible and acts as a spring, we performed immunohistochemistry to test whether changes to this protein could have an effect on sarcomere length. An antibody to α-actinin was used to demarcate Z-lines in longitudinal sections of the tibialis anterior muscle and sarcomere length was measured as the distance between Z-lines. While average sarcomere length was not different between iMSBmal1+/+ and iMSBmal1-/- muscle, variation in sarcomere length increased following Bmal1 knockout. To test if increased variability of titin isoform expression similar to the iMSBmal1-/- mouse could lead to variable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 snRNPs targeted to titin PEVK regions. Sarcomere length variability will be measured in these cells following 7 days of differentation. Current studies with this model are ongoing and will lead to increased knowledge of titin's role in regulating sarcomere length in a skeletal muscle at rest.

TDRD6 mediates early steps of spliceosome maturation in primary spermatocytes.

Tudor containing protein 6 (TDRD6) is a male germ line-specific protein essential for chromatoid body (ChB) structure, elongated spermatid development and male fertility. Here we show that in meiotic prophase I spermatocytes TDRD6 interacts with the key protein arginine methyl transferase PRMT5, which supports splicing. TDRD6 also associates with spliceosomal core protein SmB in the absence of RNA and in an arginine methylation dependent manner. In Tdrd6-/- diplotene spermatocytes PRMT5 association with SmB and arginine dimethylation of SmB are much reduced. TDRD6 deficiency impairs the assembly of spliceosomes, which feature 3.5-fold increased levels of U5 snRNPs. In the nucleus, these deficiencies in spliceosome maturation correlate with decreased numbers of SMN-positive bodies and Cajal bodies involved in nuclear snRNP maturation. Transcriptome analysis of TDRD6-deficient diplotene spermatocytes revealed high numbers of splicing defects such as aberrant usage of intron and exons as well as aberrant representation of splice junctions. Together, this study demonstrates a novel function of TDRD6 in spliceosome maturation and mRNA splicing in prophase I spermatocytes.

Will the circle be unbroken: specific mutations in the yeast Sm protein ring expose a requirement for assembly factor Brr1, a homolog of Gemin2.

A seven-subunit Sm protein ring assembles around specific U-rich RNA segments of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP crystal structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmD1 and SmD2 subunits. Of 18 conserved amino acids comprising their RNA-binding sites or intersubunit interfaces, only Arg88 in SmD1 and Arg97 in SmD2 were essential for growth. Tests for genetic interactions with non-Sm splicing factors identified benign mutations of SmD1 (N37A, R88K, R93A) and SmD2 (R49A, N66A, R97K, D99A) that were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and/or Msl1. Tests of 264 pairwise combinations of SmD1 and SmD2 alleles with each other and with a collection of SmG, SmE, SmF, SmB, and SmD3 alleles revealed 92 instances of inter-Sm synthetic lethality. We leveraged the Sm mutant collection to illuminate the function of the yeast Sm assembly factor Brr1 and its relationship to the metazoan Sm assembly factor Gemin2. Mutations in the adjacent SmE (K83A), SmF (K32A, F33A, R74K), SmD2 (R49A, N66A, E74A, R97K, D99A), and SmD1 (E18A, N37A) subunits—but none in the SmG, SmD3, and SmB subunits—were synthetically lethal with brr1Δ. Using complementation of brr1Δ lethality in two Sm mutant backgrounds as an in vivo assay of Brr1 activity, we identified as essential an N-terminal segment of Brr1 (amino acids 24–47) corresponding to the Gemin2 α1 helix that interacts with SmF and a Brr1 C-terminal peptide (336QKDLIE341) that, in Gemin2, interacts with SmD2.

Changes in the detergent-insoluble brain proteome linked to amyloid and tau in Alzheimer's Disease progression.

Despite a key role of amyloid-beta (Aβ) in Alzheimer's disease (AD), mechanisms that link Aβ plaques to tau neurofibrillary tangles and cognitive decline still remain poorly understood. The purpose of this study was to quantify proteins in the sarkosyl-insoluble brain proteome correlated with Aβ and tau insolubility in the asymptomatic phase of AD (AsymAD) and through mild cognitive impairment (MCI) and symptomatic AD. Employing label-free mass spectrometry-based proteomics, we quantified 2711 sarkosyl-insoluble proteins across the prefrontal cortex from 35 individual cases representing control, AsymAD, MCI and AD. Significant enrichment of Aβ and tau in AD was observed, which correlated with neuropathological measurements of plaque and tau tangle density, respectively. Pairwise correlation coefficients were also determined for all quantified proteins to Aβ and tau, across the 35 cases. Notably, six of the ten most correlated proteins to Aβ were U1 small nuclear ribonucleoproteins (U1 snRNPs). Three of these U1 snRNPs (U1A, SmD and U1-70K) also correlated with tau consistent with their association with tangle pathology in AD. Thus, proteins that cross-correlate with both Aβ and tau, including specific U1 snRNPs, may have potential mechanistic roles in linking Aβ plaques to tau tangle pathology during AD progression.

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators.

Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery.

Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

ABSTRACTCo-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.

Re-refinement of the spliceosomal U4 snRNP core-domain structure.

The core domain of small nuclear ribonucleoprotein (snRNP), comprised of a ring of seven paralogous proteins bound around a single-stranded RNA sequence, functions as the assembly nucleus in the maturation of U1, U2, U4 and U5 spliceosomal snRNPs. The structure of the human U4 snRNP core domain was initially solved at 3.6 Å resolution by experimental phasing using data with tetartohedral twinning. Molecular replacement from this model followed by density modification using untwinned data recently led to a structure of the minimal U1 snRNP at 3.3 Å resolution. With the latter structure providing a search model for molecular replacement, the U4 core-domain structure has now been re-refined. The U4 Sm site-sequence AAUUUUU has been shown to bind to the seven Sm proteins SmF-SmE-SmG-SmD3-SmB-SmD1-SmD2 in an identical manner as the U1 Sm-site sequence AAUUUGU, except in SmD1 where the bound U replaces G. The progression from the initial to the re-refined structure exemplifies a tortuous route to accuracy: where well diffracting crystals of complex assemblies are initially unavailable, the early model errors are rectified by exploiting preliminary interpretations in further experiments involving homologous structures. New insights are obtained from the more accurate model.

Crystallization and biochemical characterization of the human spliceosomal Aar2-Prp8(RNaseH) complex.

In eukaryotes, the removal of nuclear noncoding sequences (pre-mRNA splicing) is catalyzed by the spliceosome, which consists of five ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs, each with a respective snRNA) and a plethora of protein factors that aid spliceosomal maturation, assembly, activation and disassembly. Recently, the U5 snRNP maturation factor Aar2p from baker's yeast has been characterized structurally and biochemically. Aar2p binds to the RNaseH (RH) and Jab1/MPN domains of the highly conserved U5-specific Prp8p, which forms a framework for the spliceosomal catalytic centre. Thereby, Aar2p sterically excludes Brr2p, a helicase essential for the catalytic activation of the spliceosome, from Prp8p binding. At the same time, Aar2p blocks U4/U6 di-snRNA binding to Prp8p. Aar2p therefore prevents premature spliceosome activation and its functions are regulated by reversible phosphorylation. To date, little is known about the hypothetical human Aar2 (hsAar2) orthologue C20ORF4. This study identifies C20ORF4 (i) as part of the HeLa proteome by Western blotting and (ii) as a true Aar2 orthologue which binds to the RH domain (hsRH) of Prp8 and corroborates an evolutionary link between yeast and human Aar2 function. An elaborate strategy was devised to crystallize hsAar2 in complex with hsRH. The analysis of initial weakly diffracting crystals obtained by in situ proteolysis and homology modelling guided the design of an hsAar2 construct in which an internal loop was replaced by three serines (hsAar2(Δloop)). A complex of hsAar2(Δloop) and hsRH crystallized in space group C2; the crystals diffracted to 2.35 Å resolution and were suitable for structure determination by molecular-replacement approaches. The study presented here suggests a connection between Aar2 and the spliceosome in human cells and paves the way for structural studies of human Aar2.