half pint Regulates Alternative Splice Site Selection in Drosophila

Buskirk, Cheryl Van; Schüpbach, Trudi

doi:10.1016/s1534-5807(02)00128-4

Cited by 79 publications

(95 citation statements)

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“…Though a definitive role of Otu in regulating Grk expression has not been previously described, Van Buskirk et al (Van Buskirk et al, 2002) found that four copies of Otu104 are able to rescue the grk RNA mislocalization defect of hfp mutants. Our analysis of the hypomorphic alleles, otu 7 and otu 11 , reveals a requirement for Otu in localizing grk RNA for proper DV patterning.…”

Section: Otu Plays a Role In Grk Rna Localization And Interacts With mentioning

confidence: 82%

“…After washing, the bands were visualized by the ECLPlus chemiluminescent system (Amersham). Germarial western analysis was performed according to Van Buskirk et al (Van Buskirk et al, 2002) with 20 germaria in 50 µl of loading buffer (5 M Urea, 0.125 M Tris [pH 6.8], 4% SDS, 10% β-mercaptoethanol, 20% glycerol and 0.1% Bromophenol Blue). Samples were loaded on a 7% NuPAGE Tris-Acetate pre-cast gel (Invitrogen), transferred, blocked, probed and detected as described above using guinea pig anti-Otu (1:500 in TBST + 5% milk) or monoclonal anti-Tubulin (1:250 in TBST + 5% milk; Sigma T-9026).…”

Section: Generation Of Sqd Antibodies Immunoprecipitations and Westementioning

confidence: 99%

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Hrb27C, Sqd and Otu cooperatively regulategurkenRNA localization and mediate nurse cell chromosome dispersion inDrosophilaoogenesis

2004

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Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophilaoogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. We show that Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. Our data demonstrate a novel role for Hrb27C in promoting grk localization. We also observe a direct physical interaction between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. We find that some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grklocalization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. Our analyses support dual cooperative roles for Sqd,Hrb27C and Otu during Drosophila oogenesis.

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Section: Otu Plays a Role In Grk Rna Localization And Interacts With mentioning

confidence: 82%

Section: Generation Of Sqd Antibodies Immunoprecipitations and Westementioning

confidence: 99%

Hrb27C, Sqd and Otu cooperatively regulategurkenRNA localization and mediate nurse cell chromosome dispersion inDrosophilaoogenesis

2004

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“…Probably because of a similar mechanism, mutations in the Drosophila homolog of Puf60, Hfp (Half Pint), lead to increased expression of d-myc genes, thus negatively regulating cell cycle progression (30). Hfp mutations also lead to aberrant splicing of specific mRNAs in Drosophila ovaries (31). Similar to its mammalian ortholog Puf60, Hfp is thus a regulator both of transcription and of alternative splicing.…”

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confidence: 99%

Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60

Corsini

Hothorn

Stier

et al. 2009

Journal of Biological Chemistry

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PUF60 is an essential splicing factor functionally related and homologous to U2AF65 . Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophancontaining linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 Å resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF 65 , and SF3b155. Compared with U2AF 65 -UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF 65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.Pre-mRNA splicing is a stepwise process initiated by the recognition of sequence elements at the splice site by specific splicing factors (1). The branch point sequence is recognized by splicing factor SF1 (2, 3), whereas the polypyrimidine tract and the 3Ј splice site AG-dinucleotide are bound by the heterodimer U2AF 65 -U2AF 35 (4 -7). Although SF1 alone interacts only weakly with the branch point sequence, this interaction is stabilized significantly by U2AF 65 , which binds simultaneously to SF1 and to the polypyrimidine tract (8). In the next step of splicing initiation U2 snRNP is brought to the 3Ј splice site. This involves base pairing of the U2 RNA to the branch site RNA (9) and localization of the SF3b subunit p14 near the branch point adenosine by an interaction with the N terminus of the U2 snRNP component . Initial contacts between the U2 snRNP and the pre-mRNA are mediated by the N terminus of SF3b155 binding to U2AF 65 and displacing SF1 from the branch point sequence (13).The -UHM/ SF1-ULM (15), and the UHM of SPF45 (splicing factor 45 kDa) bound to an ULM in SF3b155 (23) all share a very similar mode of molecular recognition, suggesting that UHMs in other proteins might bind similar linear motifs as well. Because the ULM consensus motif is rather short ((K/R) 4 -6 X 0 -1

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“…The interaction between Hay and Hfp is of particular interest as, like Hay/XPB, Hfp has been implicated as a dual function protein. Initially, due to the presence of the RNA recognition motif it was implicated in RNA splicing in Drosophila 19,67 . However, our work suggests Hfp also behaves as a dMYC transcriptional repressor, likely via binding singlestranded DNA to maintain RNA Pol II pausing on the dMYC promoter.…”

Section: Discussionmentioning

confidence: 99%

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

et al. 2015

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Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.

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half pint Regulates Alternative Splice Site Selection in Drosophila

Cited by 79 publications

References 50 publications

Hrb27C, Sqd and Otu cooperatively regulategurkenRNA localization and mediate nurse cell chromosome dispersion inDrosophilaoogenesis

Hrb27C, Sqd and Otu cooperatively regulategurkenRNA localization and mediate nurse cell chromosome dispersion inDrosophilaoogenesis

Dimerization and Protein Binding Specificity of the U2AF Homology Motif of the Splicing Factor Puf60

Defective Hfp-dependent transcriptional repression of dMYC is fundamental to tissue overgrowth in Drosophila XPB models

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