Yutaka Muto

Transcription mediated by RNA polymerase II (RNAPII) involves multiple stages, including initiation, promoter-proximal pausing for capping, elongation, and termination. The C-terminal domain of RNAPII (CTD) contains repetitions of the heptad consensus sequence, such as Y₁S₂P₃T₄S₅P₆S₇ with some variety, and the phosphorylated positions in the heptad sequences are altered according to the transcriptional stages. The interaction between several regulatory protein factors and the phosphorylated heptad sequence plays an important role in the accurate progress of transcription. A subset of these regulatory proteins possesses a CTD-interacting domain (CID) that specifically recognizes the phosphorylated CTD and mediates stage-specific transcriptional control. Among them, SCAF8 (RBM16), which also contains a CID, plays a key role in accurate transcriptional termination in conjunction with its paralog SCAF4. Despite their importance, the precise molecular mechanisms through which SCAF8 and SCAF4 coordinate transcriptional termination via their CID domains remain poorly understood. In this study, we report the ¹H, ¹⁵N, and ¹³C NMR resonance assignments and solution structure of the human SCAF8 CID domain. The structure exhibits an α1-α2-α3-α4-α5-α6-α7-α8 helical topology, consistent with the previously determined crystal structure. These assignments provide a valuable foundation for understanding how SCAF8 interacts with the RNAPII CTD and contributes to transcriptional elongation and termination.

The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the ¹H, ¹⁵N, and ¹³C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1–α2–α3–α4–α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–3₁₀–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.

Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5′-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the ¹H, ¹⁵N, and ¹³C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1–β1–β2–α2(kinked)–β3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and β1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis.

Matrin-3 is a multifunctional protein that binds to both DNA and RNA. Its DNA-binding activity is linked to the formation of the nuclear matrix and transcriptional regulation, while its RNA-binding activity is linked to mRNA metabolism including splicing, transport, stabilization, and degradation. Correspondingly, Matrin-3 has two zinc finger domains for DNA binding and two consecutive RNA recognition motif (RRM) domains for RNA binding. Matrin-3 has been reported to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) when its disordered region contains pathogenic mutations. Simultaneously, it has been shown that the RNA-binding activity of Matrin-3 mediated by its RRM domains, affects the formation of insoluble cytoplasmic granules, which are related to the pathogenic mechanism of ALS/FTD. Thus, the effect of the RRM domains on the phase separation of condensed protein/RNA mixtures has to be clarified for a comprehensive understanding of ALS/FTD. Here, we report the ¹H, ¹⁵N, and ¹³C resonance assignments of the two RNA binding domains and their solution structures. The resonance assignments and the solution structures obtained in this work will contribute to the elucidation of the molecular basis of Matrin-3 in the pathogenic mechanism of ALS and/or FTD.

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m⁶A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m⁶A differs between these proteins. Here, we report ¹H, ¹³C and ¹⁵N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a β1–α1–β2–α2–β3–β4–β5–α3–β6–α4 topology, which was also observed in YTH1. However, the β4–β5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m⁶A-binding pocket of YTH2 formed by the β4–β5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.

The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598–631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of β1‐α1‐β2‐β3‐α2‐β4. Furthermore, a docking model based on NOESY measurements suggests that residues 607–616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix–helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull‐down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix–helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.

The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3′ splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the β sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.