Crystal Structures and RNA-binding Properties of the RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein L: INSIGHTS INTO ITS ROLES IN ALTERNATIVE SPLICING REGULATION

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.     

The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids

TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved β-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.     

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information.     

Functional stabilization of an RNA recognition motif by a noncanonical N-terminal expansion

RNA recognition motifs (RRMs) constitute versatile macromolecular interaction platforms. They are found in many components of spliceosomes, in which they mediate RNA and protein interactions by diverse molecular strategies. The human U11/U12-65K protein of the minor spliceosome employs a C-terminal RRM to bind hairpin III of the U12 small nuclear RNA (snRNA). This interaction comprises one side of a molecular bridge between the U11 and U12 small nuclear ribonucleoprotein particles (snRNPs) and is reminiscent of the binding of the N-terminal RRMs in the major spliceosomal U1A and U2B″ proteins to hairpins in their cognate snRNAs. Here we show by mutagenesis and electrophoretic mobility shift assays that the β-sheet surface and a neighboring loop of 65K C-terminal RRM are involved in RNA binding, as previously seen in canonical RRMs like the N-terminal RRMs of the U1A and U2B″ proteins. However, unlike U1A and U2B″, some 30 residues N-terminal of the 65K C-terminal RRM core are additionally required for stable U12 snRNA binding. The crystal structure of the expanded 65K C-terminal RRM revealed that the N-terminal tail adopts an α-helical conformation and wraps around the protein toward the face opposite the RNA-binding platform. Point mutations in this part of the protein had only minor effects on RNA affinity. Removal of the N-terminal extension significantly decreased the thermal stability of the 65K C-terminal RRM. These results demonstrate that the 65K C-terminal RRM is augmented by an N-terminal element that confers stability to the domain, and thereby facilitates stable RNA binding.     

A Compare-and-Contrast NMR Dynamics Study of Two Related RRMs: U1A and SNF

The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/β sandwich topology, and these RRMs use their β-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the β-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 β-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.     

Distinct functions of the closely related tandem RNA-recognition motifs of hnRNP A1.

hnRNP A1 regulates alternative splicing by antagonizing SR proteins. It consists of two closely related, tandem RNA-recognition motifs (RRMs), followed by a glycine-rich domain. Analysis of variant proteins with duplications, deletions, or swaps of the RRMs showed that although both RRMs are required for alternative splicing function, each RRM plays distinct roles, and their relative position is important. Surprisingly, RRM2 but not RRM1 could support this function when duplicated, despite their very similar structure. Specific RNA binding and annealing are not sufficient for hnRNP A1 alternative splicing function. These observations, together with phylogenetic and structural data, suggest that the two RRMs are quasi-symmetric but functionally nonequivalent modules that evolved as components of a single bipartite domain.     

Human UP1 as a model for understanding purine recognition in the family of proteins containing the RNA recognition motif (RRM)

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is a prototype for the family of eukaryotic RNA processing proteins containing the common RNA recognition motif (RRM). The region consisting of residues 1-195 of hnRNP A1 is referred to as UP1. This region has two RRMs and has a high affinity for both single-stranded RNA and the human telomeric repeat sequence d(TTAGGG)(n). We have used UP1's novel DNA binding to investigate how RRMs bind nucleic acid bases through their highly conserved RNP consensus sequences. Nine complexes of UP1 bound to modified telomeric repeats were investigated using equilibrium fluorescence binding and X-ray crystallography. In two of the complexes, alteration of a guanine to either 2-aminopurine or nebularine resulted in an increase in K(d) from 88nM to 209nM and 316nM, respectively. The loss of these orienting interactions between UP1 and the substituted base allows it to flip between syn and anti conformations. Substitution of the same base with 7-deaza-guanine preserves the O6/N1 contacts but still increases the K(d) to 296nM and suggests that it is not simply the loss of affinity that gives rise to the base mobility, but also the stereochemistry of the specific contact to O6. Although these studies provide details of UP1 interactions to nucleic acids, three general observations about RRMs are also evident: (1) as suggested by informatic studies, main-chain to base hydrogen bonding makes up an important aspect of ligand recognition (2) steric clashes generated by modification of a hydrogen bond donor-acceptor pair to a donor-donor pair are poorly tolerated and (3) a conserved lysine position proximal to RNP-2 (K(106)-IFVGGI) orients the purine to allow stereochemical discrimination between adenine and guanine based on the 6-position. This single interaction is well-conserved in known RRM structures and appears to be a broad indicator for purine preference in the larger family of RRM proteins.     

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA, telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of mRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem betaalpha betabeta alphabeta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.     

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.     

Alternative conformations at the RNA-binding surface of the N-terminal U2AF(65) RNA recognition motif

The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF(65) variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF(65). Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1) is described at 1.47 A resolution in the absence of RNA. Notably, RNA-binding by U2AF(65) selectively stabilizes pre-existing alternative conformations of three side-chains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the beta2/beta3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF(65) to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.     

Covariance analysis of RNA recognition motifs identifies functionally linked amino acids.

The RNA recognition motif (RRM) is one of the most common eukaryotic protein motifs. RRM sequences form a conserved globular structure known as the RNA-binding domain (RBD) or the ribonucleoprotein domain. Many proteins that contain RRM sequences bind RNA in a sequence-specific manner. To investigate the basis for the RNA-binding specificity of RRMs, we subjected 330 aligned RRM sequences to covariance analysis. The analysis revealed a single network of covariant amino acid pairs comprising the buried core of the RBD and a surface patch. Structural studies have implicated a subset of these residues in RNA binding. The covariance linkages identify a larger set of amino acid residues, including some not directly in contact with bound RNA, that may influence RNA-binding specificity.     

Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.     

Recognition of polyadenylate RNA by the poly(A)-binding protein.

The cocrystal structure of human poly(A)-binding protein (PABP) has been determined at 2.6 A resolution. PABP recognizes the 3' mRNA poly(A) tail and plays critical roles in eukaryotic translation initiation and mRNA stabilization/degradation. The minimal PABP used in this study consists of the N-terminal two RRM-type RNA-binding domains connected by a short linker (RRM1/2). These two RRMs form a continuous RNA-binding trough, lined by an antiparallel beta sheet backed by four alpha helices. The polyadenylate RNA adopts an extended conformation running the length of the molecular trough. Adenine recognition is primarily mediated by contacts with conserved residues found in the RNP motifs of the two RRMs. The convex dorsum of RRM1/2 displays a phylogenetically conserved hydrophobic/acidic portion, which may interact with translation initiation factors and regulatory proteins.     

Gbp1p, a Protein with RNA Recognition Motifs, Binds Single-Stranded Telomeric DNA and Changes Its Binding Specificity upon Dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

Function of RRM domains of Drosophila melanogaster ELAV: Rnp1 mutations and rrm domain replacements with ELAV family proteins and SXL

Members of the ELAV family of proteins contain three RNA recognition motifs (RRMs), which are highly conserved. ELAV, a Drosophila melanogaster member of this family, provides a vital function and exhibits a predominantly nuclear localization. To investigate if the RNA-binding property of each of the ELAV RRMs is required for ELAV's in vivo function, amino acid residues critical in RNA binding for each RRM were individually mutated. A stringent genetic complementation test revealed that when the mutant protein was the sole source of ELAV, RNA-binding ability of each RRM was essential to ELAV function. To assess the degree to which each domain was specific for ELAV function and which domains perhaps performed a function common to related ELAV proteins, we substituted an ELAV RRM with the corresponding RRM from RBP9, the D. melanogaster protein most homologous to ELAV; HuD, a human ELAV family protein; and SXL, which, although evolutionarily related, is not an ELAV family member. This analysis revealed that RRM3 replacements were fully functional, but RRM1 and RRM2 replacements were largely nonfunctional. Under less stringent conditions RRM1 and RRM2 replacements from SXL and RRM1 replacement from RBP9 were able to provide supplemental function in the presence of a mutant hypomorphic ELAV protein.     

The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.     

The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site

Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.     

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.     

Interactions between PTB RRMs Induce Slow Motions and Increase RNA Binding Affinity

Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site-mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA recognition motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. ¹⁵N NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this time scale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high-affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation.