Engineering splicing factors with designed specificities. Nat Methods

Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Nature Methods (Impact Factor: 32.07). 10/2009; 6(11):825-30. DOI: 10.1038/nmeth.1379
Source: PubMed


Alternative splicing is generally regulated by trans-acting factors that specifically bind pre-mRNA to activate or inhibit the splicing reaction. This regulation is critical for normal gene expression, and dysregulation of splicing is closely associated with human diseases. Here we engineered artificial splicing factors by combining sequence-specific RNA-binding domains of human Pumilio1 with functional domains that regulate splicing. We applied these factors to modulate different types of alternative splicing in selected targets, to examine the activity of effector domains from natural splicing factors and to modulate splicing of an endogenous human gene, Bcl-X, an anticancer target. The designer factor targeted to Bcl-X increased the amount of pro-apoptotic Bcl-xS splice isoform, thus promoting apoptosis and increasing chemosensitivity of cancer cells to common antitumor drugs. Our approach permitted the creation of artificial factors to target virtually any pre-mRNA, providing a strategy to study splicing regulation and to manipulate disease-associated splicing events.

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Available from: Zefeng Wang, Jan 30, 2014
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    • "Compared with cognate PBSs, we observed a diminished activity towards PBSs with 1 or 2 mismatches, though some cross-reactivity is evident (Figure 2b). These observations are consistent with the cross-reactivity between WT and PUF (1SE) in in vitro assays [16] and similar cross-reactivity between WT and other mutant PUF proteins [16,25] that differ by 1-2 repeats. Overall, luciferase repression by the TPUF constructs was sequence-specific, corroborating the validity of the TPUF-reporter system. "
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    ABSTRACT: Due to their modular repeat structure, Pumilio/fem-3 mRNA binding factor (PUF) proteins are promising candidates for designer RNA-binding protein (RBP) engineering. To further facilitate the application of the PUF domain for the sequence-specific RBP engineering, a rapid cloning approach is desirable that would allow efficient introduction of multiple key amino acid mutations in the protein. Here, we report the implementation of the Golden Gate cloning method for an efficient one-step assembly of a designer PUF domain for RNA specificity engineering. We created a repeat module library that is potentially capable of generating a PUF domain with any desired specificity. PUF domains with multiple repeat modifications for the recognition of altered RNA targets were obtained in a one-step assembly reaction, which was found to be highly efficient. The new PUF variants exhibited high in vitro binding efficiencies to cognate RNA sequences, corroborating the applicability of the modular approach for PUF engineering. To demonstrate the application of the PUF domain assembly method for RBP engineering, we fused the PUF domain to a post-transcriptional regulator and observed a sequence-specific reporter and endogenous gene repression in human cell lines. The Golden Gate based cloning approach thus should allow greater flexibility and speed in implementing the PUF protein scaffold for engineering designer RBPs, and facilitate its use as a tool in basic and applied biology and medicine.
    Journal of Biological Engineering 03/2014; 8(1):7. DOI:10.1186/1754-1611-8-7 · 2.48 Impact Factor
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    • "expression vector (Invitrogen). Five hundred nanograms of splicing reporter (pZW2C‐A6G) containing the PUF3‐2 recognition sequence (Wang et al, 2009) was transfected alone, or cotransfected with 100 ng of RBM10‐PUF or PUF expression vector, respectively into HEK293T cells in 12‐well plate. After RT‐PCR, the expression level of the two isoforms including or excluding the cassette exons was measured by Bioanalyser DNA 1000 chip (Agilent). "
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    ABSTRACT: RBM10 encodes an RNA binding protein. Mutations in RBM10 are known to cause multiple congenital anomaly syndrome in male humans, the TARP syndrome. However, the molecular function of RBM10 is unknown. Here we used PAR-CLIP to identify thousands of binding sites of RBM10 and observed significant RBM10-RNA interactions in the vicinity of splice sites. Computational analyses of binding sites as well as loss-of-function and gain-of-function experiments provided evidence for the function of RBM10 in regulating exon skipping and suggested an underlying mechanistic model, which could be subsequently validated by minigene experiments. Furthermore, we demonstrated the splicing defects in a patient carrying an RBM10 mutation, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10-mediated splicing regulation and provided a molecular link to understanding a human congenital disorder.
    EMBO Molecular Medicine 09/2013; 5(9):1431-42. DOI:10.1002/emmm.201302663 · 8.67 Impact Factor
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    • "Interestingly, this transcription unit responded positively to SRSF2 overexpression and negatively to SRSF2 downregulation by RNAi, but not to SRSF1 overexpression, although we detected some effect with SRSF1 RNAi. We further tested the response of the HSV-based reporter to both of the SR proteins using a tethering approach by engineering a tandem repeat of RNA elements that can be recognized by a specific Pumilio 1 (PUF) RNA-binding motif (RRM) and by scoring the reporter response to individual SR proteins fused to the PUF RRM (Wang et al., 2009). We found that tethered (E) Restoration of RNAP II Ser2 phosphorylation in MEFs depleted of both SRSF2 and 7SK RNA. "
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    ABSTRACT: RNAP II is frequently paused near gene promoters in mammals, and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR-splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes.
    Cell 05/2013; 153(4):855-68. DOI:10.1016/j.cell.2013.04.028 · 32.24 Impact Factor
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