UBL5 is essential for pre‐mRNA splicing and sister chromatid cohesion in human cells

UBL5 is an atypical ubiquitin‐like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre‐mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre‐mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre‐mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.     

AtBUD13 affects pre‐mRNA splicing and is essential for embryo development in Arabidopsis

Pre‐mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud‐site selection protein 13) regulates the budding pattern and pre‐mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T‐DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA‐seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre‐mRNA splicing.     

Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast

Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80%. Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/MTR2 produces at least five different spliced mRNAs. One gene (YGR225W/SPO70) has an intron whose removal is activated during meiosis under control of the MER1 gene. We found eight new introns, suggesting that numerous introns still remain to be discovered. The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast.     

Structural basis for the function of DEAH helicases

DEAH helicases participate in pre‐messenger RNA splicing and ribosome biogenesis. The structure of yeast Prp43p‐ADP reveals the homology of DEAH helicases to DNA helicases and the presence of an oligonucleotide‐binding motif. A β‐hairpin from the second RecA domain is wedged between two carboxy‐terminal domains and blocks access to the occluded RNA binding site formed by the RecA domains and a C‐terminal domain. ATP binding and hydrolysis are likely to induce conformational changes in the hairpin that are important for RNA unwinding or ribonucleoprotein remodelling. The structure of Prp43p provides the framework for functional and genetic analysis of all DEAH helicases.     

nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation,is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self‐splicing ribozyme and its own intron‐encoded maturase protein. A hallmark of maturases is that they are intron‐specific, acting as cofactors that bind their intron‐containing pre‐RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron‐encoded maturase open reading frames suggest that their splicing in vivo is assisted by ‘trans’‐acting protein factors. Interestingly, angiosperms harbor several nuclear‐encoded maturase‐related (nMat) genes that contain N‐terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.     

Solution structure of the RNA binding domain in the human muscleblind‐like protein 2

The muscleblind‐like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1–4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre‐mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH‐type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β‐sheet with the N‐terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three‐dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH‐type zinc finger motifs in the MBNL protein family.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

Specific splicing defects in S. pombe carrying a degron allele of the Survival of Motor Neuron gene

Spinal muscular atrophy results from deletions or mutations in the survival of motor neuron (SMN1) gene. The SMN protein has an essential role in the biogenesis of spliceosomal snRNPs, but the link between a defect in this process and specific splicing inhibition of pre‐mRNAs has not been established. In this study, we report the construction of a temperature‐degron (td) allele of the Schizosaccharomyces pombe SMN protein and show that its depletion at 37°C affects splicing and formation of U1, U2, U4 and U5 snRNPs, but not of U6 and U3 ribonucleoproteins. The function of the tdSMN allele in snRNP assembly is already perturbed at 25°C, suggesting a deleterious effect of the tag at this temperature. Using a genome‐wide approach, we report that introns react unequally to lower levels of snRNPs in tdSMN cells and that increasing the length of the polypyrimidine tract can improve the splicing efficiency of some, but not all, affected introns. Altogether, our results suggest that the defects observed in tdSMN fission yeast cells mimic splicing deficits observed in SMN‐deficient metazoan cells.     

The Ubp6 family of deubiquitinating enzymes contains a ubiquitin-like domain: SUb.

A sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin-specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1-3) present in sequence databases. An N-terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving alpha-linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N-terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin-like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.     

NEMO specifically recognizes K63‐linked poly‐ubiquitin chains through a new bipartite ubiquitin‐binding domain

An important property of NEMO, the core element of the IKK complex involved in NF‐κB activation, resides in its ability to specifically recognize poly‐ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C‐terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63‐linked chains, we demonstrate that together they form a bipartite high‐affinity K63‐specific ubiquitin‐binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C‐terminal half of NEMO is to specifically bind K63‐linked poly‐ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly‐ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.     

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.     

Structural and functional studies of USP20 ZnF‐UBP domain by NMR

Deubiquitinase USP20/VDU2 has been demonstrated to play important roles in multiple cellular processes by controlling the life span of substrate proteins including hypoxia‐inducible factor HIF1α, and so forth. USP20 contains four distinct structural domains including the N‐terminal zinc‐finger ubiquitin binding domain (ZnF‐UBP), the catalytic domain (USP domain), and two tandem DUSP domains, and none of the structures for these four domains has been solved. Meanwhile, except for the ZnF‐UBP domain, the biological functions for USP20's catalytic domain and tandem DUSP domains have been at least partially clarified. Here in this study, we determined the solution structure of USP20 ZnF‐UBP domain and investigated its binding properties with mono‐ubiquitin and poly‐ubiquitin (K48‐linked di‐ubiquitin) by using NMR and molecular modeling techniques. USP20's ZnF‐UBP domain forms a spherically shaped fold consisting of a central β‐sheet with either one α‐helix or two α‐helices packed on each side of the sheet. However, although having formed a canonical core structure essential for ubiquitin recognition, USP20 ZnF‐UBP presents weak ubiquitin binding capacity. The structural basis for understanding USP20 ZnF‐UBP's ubiquitin binding capacity was revealed by NMR data‐driven docking. Although the electrostatic interactions between D264 of USP5 (E87 in USP20 ZnF‐UBP) and R74 of ubiquitin are kept, the loss of the extensive interactions formed between ubiquitin's di‐glycine motif and the conserved and non‐conserved residues of USP20 ZnF‐UBP domain (W41, E55, and Y84) causes a significant decrease in its binding affinity to ubiquitin. Our findings indicate that USP20 ZnF‐UBP domain might have a physiological role unrelated to its ubiquitin binding capacity.     

A conditional role of U2AF in splicing of introns with unconventional polypyrimidine tracts

Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3' splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3' splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3' splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.