The Experts below are selected from a list of 501 Experts worldwide ranked by ideXlab platform

Mikko J. Frilander - One of the best experts on this subject based on the ideXlab platform.

  • The emerging role of Minor intron splicing in neurological disorders.
    Cell stress, 2018
    Co-Authors: Daniel Jutzi, Mikko J. Frilander, Maureen Veronica Akinyi, Jonas Mechtersheimer, Marc-david Ruepp
    Abstract:

    Pre-mRNA splicing is an essential step in eukaryotic gene expression. Mutations in cis-acting sequence elements within pre-mRNA molecules or trans-acting factors involved in pre-mRNA processing have both been linked to splicing dysfunction that give rise to a large number of human diseases. These mutations typically affect the major splicing pathway, which excises more than 99% of all introns in humans. However, approximately 700-800 human introns feature divergent intron consensus sequences at their 5' and 3' ends and are recognized by a separate pre-mRNA processing machinery denoted as the Minor Spliceosome. This Spliceosome has been studied less than its major counterpart, but has received increasing attention during the last few years as a novel pathomechanistic player on the stage in neurodevelopmental and neurodegenerative diseases. Here, we review the current knowledge on Minor Spliceosome function and discuss its potential pathomechanistic role and impact in neurodegeneration.

  • Mutations in the U11/U12-65K protein associated with isolated growth hormone deficiency lead to structural destabilization and impaired binding of U12 snRNA.
    RNA (New York N.Y.), 2017
    Co-Authors: Antto Juhani Norppa, Bhupendra Verma, Tuuli M. Kauppala, Harri Heikkinen, Hideo Iwai, Mikko J. Frilander
    Abstract:

    Mutations in the components of the Minor Spliceosome underlie several human diseases. A subset of patients with isolated growth hormone deficiency (IGHD) harbors mutations in the RNPC3 gene, which encodes the Minor Spliceosome-specific U11/U12-65K protein. Although a previous study showed that IGHD patient cells have defects in U12-type intron recognition, the biochemical effects of these mutations on the 65K protein have not been characterized. Here, we show that a proline-to-threonine missense mutation (P474T) and a nonsense mutation (R502X) in the C-terminal RNA recognition motif (C-RRM) of the 65K protein impair the binding of 65K to U12 and U6atac snRNAs. We further show that the nonsense allele is targeted to the nonsense-mediated decay (NMD) pathway, but in an isoform-specific manner, with the nuclear-retained 65K long-3'UTR isoform escaping the NMD pathway. In contrast, the missense P474T mutation leads, in addition to the RNA-binding defect, to a partial defect in the folding of the C-RRM and reduced stability of the full-length protein, thus reducing the formation of U11/U12 di-snRNP complexes. We propose that both the C-RRM folding defect and NMD-mediated decrease in the levels of the U11/U12-65K protein reduce formation of the U12-type intron recognition complex and missplicing of a subset of Minor introns leading to pituitary hypoplasia and a subsequent defect in growth hormone secretion.

  • Minor Spliceosome and disease.
    Seminars in Cell & Developmental Biology, 2017
    Co-Authors: Bhupendra Verma, Maureen Veronica Akinyi, Antto Juhani Norppa, Mikko J. Frilander
    Abstract:

    The U12-dependent (Minor) Spliceosome excises a rare group of introns that are characterized by a highly conserved 5' splice site and branch point sequence. Several new congenital or somatic diseases have recently been associated with mutations in components of the Minor Spliceosome. A common theme in these diseases is the detection of elevated levels of transcripts containing U12-type introns, of which a subset is associated with other splicing defects. Here we review the present understanding of Minor Spliceosome diseases, particularly those associated with the specific components of the Minor Spliceosome. We also present a model for interpreting the molecular-level consequences of the different diseases.

  • Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.
    PLoS genetics, 2017
    Co-Authors: Jens Verbeeren, Bhupendra Verma, Elina H. Niemelä, Karen Yap, Evgeniy V. Makeyev, Mikko J. Frilander
    Abstract:

    Cellular homeostasis of the Minor Spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

  • Regulation of gene expression through inefficient splicing of U12-type introns.
    RNA biology, 2014
    Co-Authors: Elina H. Niemelä, Mikko J. Frilander
    Abstract:

    U12-type introns are a rare class of nuclear introns that are removed by a dedicated U12-dependent Spliceosome and are thought to regulate the expression of their target genes owing through their slower splicing reaction. Recent genome-wide studies on the splicing of U12-type introns are now providing new insights on the biological significance of this parallel splicing machinery. The new studies cover multiple different organisms and experimental systems, including human patient cells with mutations in the components of the Minor Spliceosome, zebrafish with similar mutations and various experimentally manipulated human cells and Arabidopsis plants. Here, we will discuss the potential implications of these studies on the understanding of the mechanism and regulation of the Minor Spliceosome, as well as their medical implications.

Hunseung Kang - One of the best experts on this subject based on the ideXlab platform.

  • Structural features important for the U12 snRNA binding and Minor Spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins
    RNA biology, 2016
    Co-Authors: Su Jung Park, Hyun Ju Jung, Sy Nguyen Dinh, Hunseung Kang
    Abstract:

    Although seven proteins unique to U12 intron-specific Minor Spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the Minor Spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for Minor Spliceosome assembly.

  • structural features important for the u12 snrna binding and Minor Spliceosome assembly of arabidopsis u11 u12 small nuclear ribonucleoproteins
    RNA Biology, 2016
    Co-Authors: Su Jung Park, Hyun Ju Jung, Sy Nguyen Dinh, Hunseung Kang
    Abstract:

    Although seven proteins unique to U12 intron-specific Minor Spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the Minor Spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for Minor Spliceosome assembly.

  • The Arabidopsis homolog of human Minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development
    Journal of experimental botany, 2016
    Co-Authors: Bo Mi Kim, Hyun Ju Jung, Kyung Jin Kwak, Hunseung Kang
    Abstract:

    The Minor U12 introns are removed from precursor mRNAs by the U12 intron-specific Minor Spliceosome. Among the seven ribonucleoproteins unique to the Minor Spliceosome, denoted as U11/U12-20K, U11/U12-25K, U11/U12-31K, U11/U12-65K, U11-35K, U11-48K, and U11-59K, the roles of only U11/U12-31K and U11/U12-65K have been demonstrated in U12 intron splicing and plant development. Here, the functional role of the Arabidopsis homolog of human U11-48K in U12 intron splicing and the development of Arabidopsis thaliana was examined using transgenic knockdown plants. The u11-48k mutants exhibited several defects in growth and development, such as severely arrested primary inflorescence stems, formation of serrated leaves, production of many rosette leaves after bolting, and delayed senescence. The splicing of most U12 introns analyzed was impaired in the u11-48k mutants. Comparative analysis of the splicing defects and phenotypes among the u11/u12-31k, u11-48k, and u11/12-65k mutants showed that the severity of abnormal development was closely correlated with the degree of impairment in U12 intron splicing. Taken together, these results provide compelling evidence that the Arabidopsis homolog of human U11-48K protein, as well as U11/U12-31K and U11/U12-65K proteins, is necessary for correct splicing of U12 introns and normal plant growth and development.

  • the arabidopsis u11 u12 65k is an indispensible component of Minor Spliceosome and plays a crucial role in u12 intron splicing and plant development
    Plant Journal, 2014
    Co-Authors: Hyun Ju Jung, Hunseung Kang
    Abstract:

    The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific Minor Spliceosome. Although several proteins unique to Minor Spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of Minor Spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.

  • The Arabidopsis U11/U12-65K is an indispensible component of Minor Spliceosome and plays a crucial role in U12 intron splicing and plant development.
    The Plant journal : for cell and molecular biology, 2014
    Co-Authors: Hyun Ju Jung, Hunseung Kang
    Abstract:

    The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific Minor Spliceosome. Although several proteins unique to Minor Spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of Minor Spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.

Hyun Ju Jung - One of the best experts on this subject based on the ideXlab platform.

  • Structural features important for the U12 snRNA binding and Minor Spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins
    RNA biology, 2016
    Co-Authors: Su Jung Park, Hyun Ju Jung, Sy Nguyen Dinh, Hunseung Kang
    Abstract:

    Although seven proteins unique to U12 intron-specific Minor Spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the Minor Spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for Minor Spliceosome assembly.

  • structural features important for the u12 snrna binding and Minor Spliceosome assembly of arabidopsis u11 u12 small nuclear ribonucleoproteins
    RNA Biology, 2016
    Co-Authors: Su Jung Park, Hyun Ju Jung, Sy Nguyen Dinh, Hunseung Kang
    Abstract:

    Although seven proteins unique to U12 intron-specific Minor Spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the Minor Spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for Minor Spliceosome assembly.

  • The Arabidopsis homolog of human Minor spliceosomal protein U11-48K plays a crucial role in U12 intron splicing and plant development
    Journal of experimental botany, 2016
    Co-Authors: Bo Mi Kim, Hyun Ju Jung, Kyung Jin Kwak, Hunseung Kang
    Abstract:

    The Minor U12 introns are removed from precursor mRNAs by the U12 intron-specific Minor Spliceosome. Among the seven ribonucleoproteins unique to the Minor Spliceosome, denoted as U11/U12-20K, U11/U12-25K, U11/U12-31K, U11/U12-65K, U11-35K, U11-48K, and U11-59K, the roles of only U11/U12-31K and U11/U12-65K have been demonstrated in U12 intron splicing and plant development. Here, the functional role of the Arabidopsis homolog of human U11-48K in U12 intron splicing and the development of Arabidopsis thaliana was examined using transgenic knockdown plants. The u11-48k mutants exhibited several defects in growth and development, such as severely arrested primary inflorescence stems, formation of serrated leaves, production of many rosette leaves after bolting, and delayed senescence. The splicing of most U12 introns analyzed was impaired in the u11-48k mutants. Comparative analysis of the splicing defects and phenotypes among the u11/u12-31k, u11-48k, and u11/12-65k mutants showed that the severity of abnormal development was closely correlated with the degree of impairment in U12 intron splicing. Taken together, these results provide compelling evidence that the Arabidopsis homolog of human U11-48K protein, as well as U11/U12-31K and U11/U12-65K proteins, is necessary for correct splicing of U12 introns and normal plant growth and development.

  • the arabidopsis u11 u12 65k is an indispensible component of Minor Spliceosome and plays a crucial role in u12 intron splicing and plant development
    Plant Journal, 2014
    Co-Authors: Hyun Ju Jung, Hunseung Kang
    Abstract:

    The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific Minor Spliceosome. Although several proteins unique to Minor Spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of Minor Spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.

  • The Arabidopsis U11/U12-65K is an indispensible component of Minor Spliceosome and plays a crucial role in U12 intron splicing and plant development.
    The Plant journal : for cell and molecular biology, 2014
    Co-Authors: Hyun Ju Jung, Hunseung Kang
    Abstract:

    The U12-dependent introns have been identified in a wide range of eukaryotes and are removed from precursor-mRNAs by U12 intron-specific Minor Spliceosome. Although several proteins unique to Minor Spliceosome have been identified, the nature of their effect on U12 intron splicing as well as plant growth and development remain largely unknown. Here, we characterized the functional role of an U12-type spliceosomal protein, U11/U12-65K in Arabidopsis thaliana. The transgenic knockdown plants generated by artificial miRNA-mediated silencing strategy exhibited severe defect in growth and development, such as severely arrested primary inflorescence stems, serrated leaves, and the formation of many rosette leaves after bolting. RNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses revealed that splicing of 198 out of the 234 previously predicted U12 intron-containing genes and 32 previously unidentified U12 introns was impaired in u11/u12-65k mutant. Moreover, the U11/U12-65K mutation affected alternative splicing, as well as U12 intron splicing, of many introns. Microarray analysis revealed that the genes involved in cell wall biogenesis and function, plant development, and metabolic processes are differentially expressed in the mutant plants. U11/U12-65K protein bound specifically to U12 small nuclear RNA (snRNA), which is necessary for branch-point site recognition. Taken together, these results provide clear evidence that U11/U12-65K is an indispensible component of Minor Spliceosome and involved in U12 intron splicing and alternative splicing of many introns, which is crucial for plant development.

Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.

  • Alternative pre‐mRNA Splicing - The Spliceosome in Constitutive Splicing
    Alternative pre-mRNA Splicing, 2012
    Co-Authors: Patrizia Fabrizio, Reinhard Luhrmann
    Abstract:

    During the splicing reaction, the Spliceosome undergoes rearrangements ofRNA–RNA interactions and an exchange of protein factors.5.1IntroductionPre-mRNA splicing is catalyzed by an elaborate and dynamic multi-megadaltonribonucleoprotein (RNP) machine, termed the Spliceosome (size 4–5MDa). Mostpre-mRNA introns are removed by the U2-dependent (major) Spliceosome that isfound in all eukaryotes. The less-abundant U12-dependent (Minor) Spliceosome,on the other hand, splices a rare class of pre-mRNA introns that is found in onlya subset of eukaryotes. Here, attention will be focused on the function of the majorSpliceosome in constitutive splicing, with reference being made to human andyeast (Saccharomyces cerevisiae), in both of which the process has been extensivelycharacterized.5.2The Mechanism of SplicingWithin an assembled Spliceosome, intron removal from the pre-mRNA substrateproceeds by way of two transesterification reactions (Figure 5.1a). In the first step,the 2

  • Minor Spliceosome components are predominantly localized in the nucleus.
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Heli K. J. Pessa, Cindy L Will, Reinhard Luhrmann, Claudia Schneider, Xiaojuan Meng, Nicholas J. Watkins, Nina Perälä, Mariann Nymark, Janne J. Turunen, Mikko J. Frilander
    Abstract:

    Recently, it has been reported that there is a differential subcellular distribution of components of the Minor U12-dependent and major U2-dependent Spliceosome, and further that the Minor Spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and Minor Spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the Minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent Spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major Spliceosome, components of the U12-dependent Spliceosome are localized predominantly in the nucleus.

  • Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase
    RNA (New York N.Y.), 2006
    Co-Authors: Ralf Trippe, Reinhard Luhrmann, Elena Guschina, Markus Hossbach, Henning Urlaub, Bernd-joachim Benecke
    Abstract:

    Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the Minor Spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.

  • the human 18s u11 u12 snrnp contains a set of novel proteins not found in the u2 dependent Spliceosome
    RNA, 2004
    Co-Authors: Cindy L Will, Claudia Schneider, Markus Hossbach, Henning Urlaub, Reinhard Rauhut, Sayda M Elbashir, Thomas Tuschl, Reinhard Luhrmann
    Abstract:

    U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5 splice site and branchpoint sequence. Thus, within the U12-type preSpliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major Spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein–protein and protein–RNA interactions contributing to 5 splice site recognition and/or intron bridging appear to differ significantly in the Minor versus major preSpliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type Spliceosome provides insight into potential evolutionary relationships between the major and Minor Spliceosome.

  • The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent Spliceosome
    RNA (New York N.Y.), 2004
    Co-Authors: Cindy L Will, Claudia Schneider, Markus Hossbach, Henning Urlaub, Reinhard Rauhut, Sayda M Elbashir, Thomas Tuschl, Reinhard Luhrmann
    Abstract:

    U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5 splice site and branchpoint sequence. Thus, within the U12-type preSpliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major Spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein–protein and protein–RNA interactions contributing to 5 splice site recognition and/or intron bridging appear to differ significantly in the Minor versus major preSpliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type Spliceosome provides insight into potential evolutionary relationships between the major and Minor Spliceosome.

Cindy L Will - One of the best experts on this subject based on the ideXlab platform.

  • U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III.
    Scientific reports, 2016
    Co-Authors: Jagjit Singh, Cindy L Will, Kavleen Sikand, Heike Conrad, Anton A. Komar, Girish C. Shukla
    Abstract:

    Formation of catalytic core of the U12-dependent Spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent Spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the Minor Spliceosome are highly plastic as compared to the major Spliceosome.

  • Minor Spliceosome components are predominantly localized in the nucleus.
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Heli K. J. Pessa, Cindy L Will, Reinhard Luhrmann, Claudia Schneider, Xiaojuan Meng, Nicholas J. Watkins, Nina Perälä, Mariann Nymark, Janne J. Turunen, Mikko J. Frilander
    Abstract:

    Recently, it has been reported that there is a differential subcellular distribution of components of the Minor U12-dependent and major U2-dependent Spliceosome, and further that the Minor Spliceosome functions in the cytoplasm. To study the subcellular localization of the snRNA components of both the major and Minor Spliceosomes, we performed in situ hybridizations with mouse tissues and human cells. In both cases, all spliceosomal snRNAs were nearly exclusively detected in the nucleus, and the Minor U11 and U12 snRNAs were further shown to colocalize with U4 and U2, respectively, in human cells. Additionally, we examined the distribution of several spliceosomal snRNAs and proteins in nuclear and cytoplasmic fractions isolated from human cells. These studies revealed an identical subcellular distribution of components of both the U12- and U2-dependent Spliceosomes. Thus, our data, combined with several earlier publications, establish that, like the major Spliceosome, components of the U12-dependent Spliceosome are localized predominantly in the nucleus.

  • the human 18s u11 u12 snrnp contains a set of novel proteins not found in the u2 dependent Spliceosome
    RNA, 2004
    Co-Authors: Cindy L Will, Claudia Schneider, Markus Hossbach, Henning Urlaub, Reinhard Rauhut, Sayda M Elbashir, Thomas Tuschl, Reinhard Luhrmann
    Abstract:

    U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5 splice site and branchpoint sequence. Thus, within the U12-type preSpliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major Spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein–protein and protein–RNA interactions contributing to 5 splice site recognition and/or intron bridging appear to differ significantly in the Minor versus major preSpliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type Spliceosome provides insight into potential evolutionary relationships between the major and Minor Spliceosome.

  • The human 18S U11/U12 snRNP contains a set of novel proteins not found in the U2-dependent Spliceosome
    RNA (New York N.Y.), 2004
    Co-Authors: Cindy L Will, Claudia Schneider, Markus Hossbach, Henning Urlaub, Reinhard Rauhut, Sayda M Elbashir, Thomas Tuschl, Reinhard Luhrmann
    Abstract:

    U11 and U12 snRNPs bind U12-type pre-mRNAs as a preformed di-snRNP complex, simultaneously recognizing the 5 splice site and branchpoint sequence. Thus, within the U12-type preSpliceosome, U11/U12 components form a molecular bridge connecting both ends of the intron. We have affinity purified human 18S U11/U12 and 12S U11 snRNPs, and identified their protein components by using mass spectrometry. U11/U12 snRNPs lack all known U1 snRNP proteins but contain seven novel proteins (i.e., 65K, 59K, 48K, 35K, 31K, 25K, 20K) not found in the major Spliceosome, four of which (59K, 48K, 35K, and 25K) are U11-associated. Thus, protein–protein and protein–RNA interactions contributing to 5 splice site recognition and/or intron bridging appear to differ significantly in the Minor versus major preSpliceosome. The majority of U11/U12 proteins are highly conserved in organisms known to contain U12-type introns. However, homologs of those associated with U11 were not detected in Drosophila melanogaster, consistent with the presence of a divergent U11 snRNP in flies. RNAi experiments revealed that several U11/U12 proteins are essential for cell viability, suggesting they play key roles in U12-type splicing. The presence of unique U11/U12 snRNP proteins in the U12-type Spliceosome provides insight into potential evolutionary relationships between the major and Minor Spliceosome.

  • Identification of Both Shared and Distinct Proteins in the Major and Minor Spliceosomes
    Science, 1999
    Co-Authors: Cindy L Will, Robin Reed, Claudia Schneider, Reinhard Luhrmann
    Abstract:

    In metazoans, two distinct Spliceosomes catalyzing pre–messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the Minor (U12-dependent) Spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the Minor Spliceosome or common to both Spliceosomes. Common proteins include four U2 snRNP polypeptides that constitute the essential splicing factor SF3b. A 35-kilodalton U11-associated protein homologous to the U1 snRNP 70K protein was also identified. These data provide fundamental information about proteins of the Minor Spliceosome and shed light on its evolutionary relationship to the major Spliceosome.