Identification and Characterization of Human Genes Encoding Hprp3p and Hprp4p, Interacting Components of the Spliceosome

Wang, Anan; Forman-Kay, Julie D.; Luo, Yu; Luo, Ming; Chow, Yu‐Hua; Plumb, Jonathan; Friesen, James D.; Tsui, Lap Chee; Heng, Henry H.Q.; Woolford, John L.; Hu, Jim

doi:10.1093/hmg/6.12.2117

Cited by 31 publications

(36 citation statements)

References 48 publications

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“…This protein domain is unique in the database, and its specific function is unknown. Like PRPF31p, HPRP3 is a component of the U4/U6 snRNP (Wang et al 1997). In mammals, HPRP3 is thought to recruit HPRP4 to the U4/U6 snRNP (Gonzalez-Santos et al 2002).…”

Section: Retinitis Pigmentosamentioning

confidence: 99%

Pre-mRNA splicing and human disease

Faustino¹,

Cooper²

2003

Genes Dev.

1,127

862

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The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977;Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999;Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002). Constitutive splicing and the basal splicing machineryThe typical human gene contains an average of 8 exons. Internal exons average 145 nucleotides (nt) in length, and introns average more than 10 times this size and can be much larger (Lander et al. 2001). Exons are defined by rather short and degenerate classical splice-site sequences at the intron/exon borders (5Ј splice site, 3Ј splice site, and branch site; Fig. 1A). Components of the basal splicing machinery bind to the classical splice-site sequences and promote assembly of the multicomponent splicing complex known as the spliceosome. The spliceosome performs the two primary functions of splicing: recognition of the intron/exon boundaries and catalysis of the cut-and-paste reactions that remove introns and join exons. The spliceosome is made up of five small nuclear ribonucleoproteins (snRNPs) and >100 proteins. Each snRNP is composed of a single uridinerich small nuclear RNA (snRNA) and multiple proteins. The U1 snRNP binds the 5Ј splice site, and the U2 snRNP binds the branch site via RNA:RNA interactions between the snRNA and the pre-mRNA (Fig. 1B). Spliceosome assembly is highly dynamic in that complex rearrangements of RNA:RNA, RNA:protein, and protein:protein interactions take place within the spliceosome. Coinciding with these internal rearrangements, both splice sites are recognized multiple times by interactions with different components during the course of spliceosome assembly (for example, see Burge et al. 1999;Du and Rosbash 2002;Lallena et al. 2002;Liu 2002). The catalytic component is likely to be U6 snRNP, which joins the spliceosome as a U4/U6 · U5 tri-snRNP (Villa et al. 2002).A splicing error that adds or removes even 1 nt will disrupt the open reading frame of an mRNA; yet exons are correctly spliced from within tens of thousands of intronic nucleotides. This remarkable precision is, in part, built into the mechanism of intron removal because once...

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Section: Retinitis Pigmentosamentioning

confidence: 99%

Pre-mRNA splicing and human disease

Faustino¹,

Cooper²

2003

Genes Dev.

1,127

862

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“…Three other Ets site mutations were generated by replacing the DNA sequences near the Ets sites with synthetic DNA fragments containing the mutations. The human ESE-1 cDNA was amplified from a human intestinal cDNA library [55] by using primers 5′-GCG AAT TCA TGG CTG CAA CCT GTG AGA-3′ (forward) and 5′-CGT AAG CTT CGA GTG GTC CGT GAG TTT GGT-3′ (reverse). Mouse ESE-3 cDNA was amplified by RT-PCR using RNA isolated from 16.5d mouse embryo kidney and the primers 5′-CCG TCG ACC CTT GCA GAT CAT GAT TCT-3′ (forward) and 5′-GGG ATC CGG TTC TTC ATT GAT CAG A-3′ (reverse).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…The protein was purified with Ni-NTA agarose beads under denaturing conditions, following the manufacturer's instructions (Qiagen). The purified protein was used as antigen for the generation of antibodies in rabbits as described [55]. Nuclear extracts containing ~20 µg protein per lane were separated by electrophoresis on 10% SDS-PAGE gel and were transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA).…”

Section: Protein Expression Antibody Generation and Western-blottingmentioning

confidence: 99%

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation

Duan

Cao

et al. 2008

Cell Res

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Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in bronchial epithelial cell lines. Treatment of these cells with IL-1β and TNF-α resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-κB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-κB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in Elf3 (homologous to human ESE-1) knockout mice, the expression of the inflammatory cytokine interleukin-6 (IL-6) is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

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“…the 15.5-kDa protein (Snu13p in yeast) that binds the 5Ј-stem-loop of U4 snRNA (24 -27), the 61-kDa protein (Prp31p in yeast) (4), and the heterotrimer cyclophilin H-Hprp4p-Hprp3p complex (26 -29). We previously identified, along with others, Hprp3p and Hprp4p as homologs of the yeast U4/U6 snRNP-specific factors Prp3p and Prp4p, respectively (28,30,31). Hprp3p and Hprp4p can be isolated from HeLa cells together with the 20-kDa cyclophilin H as a heterotrimer protein complex (32).…”

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confidence: 98%

Central Region of the Human Splicing Factor Hprp3p Interacts with Hprp4p

Gonzalez-Santos¹,

Wang²,

Jones³

et al. 2002

Journal of Biological Chemistry

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Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome. Currently, little is known about the specific roles of these factors in splicing. In this study, we characterized the molecular interaction between Hprp3p and Hprp4p. Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates. By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N-or Cterminal region did not interact with Hprp4p. In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts. Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.Pre-mRNA splicing occurs in the spliceosome, a large RNAprotein complex that contains a pre-mRNA, four essential small nuclear ribonucleoprotein (snRNP) 1 particles (U1, U2, U5, and U4/U6), and numerous non-snRNP splicing factors (1-3). Each snRNP particle consists of one (U1, U2, and U5) or two (U4/U6) snRNAs complexed with a set of Sm or Sm-like proteins and several particle-specific proteins (4 -6). These snRNPs recognize conserved sequences of the pre-mRNA and assemble into a catalytically active spliceosome that catalyzes the two cleavage-ligation reactions of pre-mRNA splicing (1,2,7,8). Spliceosome assembly follows an ordered pathway through the formation of several intermediate complexes (2). First, a pre-spliceosome (A complex) is formed once U1 and U2 snRNAs (as part of the U1 and U2 snRNPs) associate with the conserved 5Ј-splice site and the branch point of the intron, respectively (9, 10). Then, U4/U6 snRNP, in which U4 and U6 snRNAs base pair over an extended complementary region, forming a Y-shaped junction, is recruited together with U5 snRNP, presumably via protein/protein interactions, to the pre-spliceosome to form the mature spliceosome (B complex) (11-13).Prior to the first catalytic step of splicing, important conformational rearrangements occur to create a catalytically active spliceosome. For example, U1 snRNA dissociates from the 5Ј-splice site, and the U4/U6 snRNA association is disrupted (14 -16), leaving the U6 snRNA free to base pair with both the U2 snRNA and the 5Ј-splice site (17). The U2 and U6 snRNAs, together with the pre-mRNA, may form the catalytic core of the s...

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Identification and Characterization of Human Genes Encoding Hprp3p and Hprp4p, Interacting Components of the Spliceosome

Cited by 31 publications

References 48 publications

Pre-mRNA splicing and human disease

Pre-mRNA splicing and human disease

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation

Central Region of the Human Splicing Factor Hprp3p Interacts with Hprp4p

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