Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing

ABSTRACTViruses promote infection by hijacking host ubiquitin machinery to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defenses and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K/E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins (RBPs) as high-confidence substrates which are not decreased in overall abundance. We focused on two RBPs, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance, and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with virus deleted for E1B55K resulted in increased interaction of hnRNP-C with viral RNA, and attenuation of viral RNA processing. These data suggest viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing, revealing an unexpected role for non-degradative ubiquitination in manipulation of cellular processes during virus infection.

Reciprocal regulation of hnRNP C and CELF2 through translation and transcription tunes splicing activity in T cells

RNA binding proteins (RBPs) frequently regulate the expression of other RBPs in mammalian cells. Such cross-regulation has been proposed to be important to control networks of coordinated gene expression; however, much remains to be understood about how such networks of cross-regulation are established and what the functional consequence is of coordinated or reciprocal expression of RBPs. Here we demonstrate that the RBPs CELF2 and hnRNP C regulate the expression of each other, such that depletion of one results in reduced expression of the other. Specifically, we show that loss of hnRNP C reduces the transcription of CELF2 mRNA, while loss of CELF2 results in decreased efficiency of hnRNP C translation. We further demonstrate that this reciprocal regulation serves to fine tune the splicing patterns of many downstream target genes. Together, this work reveals new activities of hnRNP C and CELF2, provides insight into a previously unrecognized gene regulatory network, and demonstrates how cross-regulation of RBPs functions to shape the cellular transcriptome.

Novel Fusion Genes Involving Hnrnpc and Rarg in Acute Myeloid Leukemia Mimicking Acute Promyelocytic Leukemia

The roles of Heterogeneous nuclear ribonucleoproteins(hnRNPs) in regulating tumor development and progression, either as oncogenes or as tumor suppressors, were well documented. HnRNP C is one of the members of hnRNPs,and differential expression of hnRNP C has been found in series of tumor cells. However, the role of hnRNP C in leukemia has not been reported to date. Here, we report the first novel gene fusion event between HNRNPC and retinoic acid receptor gamma (RARG) in acute myeloid leukemia mimicking acute promyelocytic leukemia. This translocation produced the HNRNPC-RARG fusion gene and its reciprocal, RARG-HNRNPC. A 43-year-old man was referred to our hospital with fever and a sore throat.Laboratory investigations revealed the following patient characteristics: (1) white blood cell count 12 × 109/L (blasts 1% and abnormal promyelocytes 86%). (2) Morphologic analysis of the bone marrow aspirate showed 86.5% microgranular atypical promyelocytes (Figure 1a, 1b). (3) Analysis from flow cytometry showed that the blasts were positive for CD33, CD13, CD45, and cMPO and negative for CD14, CD34, CD16, CD56, HLA-DR, B- or T-cell markers. Thus, the patient started all-trans retinoic acid (ATRA) treatment immediately. Afterwards, chromosomal analysis revealed 47 metaphases, and most of them were involved in t(14;17). Fluorescence in situ hybridization and RT-PCR assays did not identify the PML/RARA, NPM-RARA, PLZF-RARArearrangement. ATRA therapy lasted for 3 weeks, but no response was observed. Next, the patient received 2 cycles of induction chemotherapy until a complete response. Afterwards, he received 6 cycles of chemotherapy. Unfortunately, the leukemia relapsed 1 year later, and all treatments (including ATRA and arsenious acid) failed to produce any effects. The patient died from sepsis. To identify molecular alterations, transcriptome sequencing analysis was performed. A 213-bp RARG-HNRNPC fusion product was specifically amplified from the patient's cDNA, as predicted (Figure 1c). Sanger sequencing showed that RARG exon 9 was fused in-frame to HNRNPC exon 3(Figure 1d). The RARG 5'-region encoding the ligand-binding domain was fused to the HNRNPC3'-region, where a cluster of phosphorylation sites is located(Figure 1e). We also found a reciprocal chimeric transcript. The amplicon size of HNRNPC-RARG fusion was 186-bp (Figure 2a). Sanger sequencing demonstrated that HNRNPC exon 3 was fused in-frame to RARG exon 5 (Figures 2b). The HNRNPC 5'-region encodes an RNA recognition motif (RRM), and the segment from RARG encodes a DNA binding domain (DBD, Figure 2c). HnRNP C ubiquitously expressed RNA-binding protein (RBP) which are believed to influence pre-mRNA metabolism such as splicing, polyadenylation, stability, transport, andtranslation mediated by internal ribosome entry site. HnRNP C also plays an essential role in cell progression and the regulation of several DNA repair proteins. Retinoic acid receptors (RARs) are transcription factors that belong to the nuclear hormone receptor family.RARA, RARB, and RARG are three RARs subtypes which share highly similar sequences and functions. A study showed RARG seems to act as a major regulator maintaining the balance between HSC self-renewal and differentiation. Acute myeloid leukemias mimicking acute promyelocytic leukemia, or acute promyelocytic-like leukemias (APLL), share the same morphology and immunocytochemistry features with typical acute promyelocytic leukemia (APL) except the RARA rearrangements, and little is known about the molecular mechanisms of APLL. The sequences and function of the RARG and RARA are highly alike, and therefore can logically explain the similarity of biological characteristics between the two entities. Three other fusion genes harboring RARG ( including NUP98-RARG , PML-RARG and CPSF6-RARG) have been found in APLL. Unfortunately they showed resistance to treatment with ATRA or ATRA plus arsenic. Moreover, poor prognosis was observed likewise. All the above confirm that RARG rearrangements are not random but recurrent genetic abnormalities. In conclusion, we present a novel HNRNPC-RARG fusion gene and its reciprocal in APLL, and suggest that at least a portion of APLLs have RARG gene rearrangements. We propose that RARG-rearranged APLL may be a novel candidate subtype of acute myelocytic leukemia, or even of APL. Disclosures No relevant conflicts of interest to declare.

Activation-Dependent TRAF3 Exon 8 Alternative Splicing Is Controlled by CELF2 and hnRNP C Binding to an Upstream Intronic Element

ABSTRACT Cell-type-specific and inducible alternative splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. We have recently shown that activation-induced skipping of TRAF3 exon 8 activates noncanonical NF-κB signaling upon T cell stimulation, but the regulatory basis for this splicing event remains unknown. Here we identify cis- and trans-regulatory elements rendering this splicing switch activation dependent and cell type specific. The cis-acting element is located 340 to 440 nucleotides upstream of the regulated exon and acts in a distance-dependent manner, since altering the location reduces its activity. A small interfering RNA screen, followed by cross-link immunoprecipitation and mutational analyses, identified CELF2 and hnRNP C as trans-acting factors that directly bind the regulatory sequence and together mediate increased exon skipping in activated T cells. CELF2 expression levels correlate with TRAF3 exon skipping in several model systems, suggesting that CELF2 is the decisive factor, with hnRNP C being necessary but not sufficient. These data suggest an interplay between CELF2 and hnRNP C as the mechanistic basis for activation-dependent alternative splicing of TRAF3 exon 8 and additional exons and uncover an intronic splicing silencer whose full activity depends on the precise location more than 300 nucleotides upstream of the regulated exon.

Regulation of CD19 Exon 2 Inclusion in B-Lymphoid Cells By Splicing Factors and Epigenetic Marks

CD19 is expressed broadly on the surface of B-cells during normal development and malignant growth, making it a good target for immunotherapy. While immunotherapies targeting CD19 have had great success against pediatric B-cell acute lymphoblastic leukemia (B-ALL), relapses lacking the CD19 epitope still occur (Maude et al., 2014). We have discovered that alternative splicing of CD19, in particular the skipping of exon 2, is responsible for the loss of CD19 extracellular domains, causing resistance to therapy (Sotillo et al., 2015). Here we investigate the molecular mechanism of CD19 exon 2 skipping. The sequence-based algorithm AVISPA (Barash et al., 2013) predicts several splicing factors (SF) to bind near exon 2. We used RNA crosslink immunoprecipitation (CLIP) in nuclear lysates from Nalm-6 B-ALL cells to test the direct binding to exon 2 of 9 AVISPA-predicted SFs and 6 SFs commonly involved in exon skipping. This allowed us to identify SRSF3, hnRNP-A, and hnRNP-C as CD19 exon 2-bound proteins. Subsequent siRNA knockdown experiments reveled that downregulation of SRSF3, but not hnRNP-C, increases the frequency of exon 2 skipping in a dose dependent manner, suggesting that SRSF3 promotes the inclusion of exon 2. To further validate the role of SRSF3 in CD19 splicing we mined the publicly available GSE52834 dataset where 22 RNA binding proteins were knocked down in the GM19238 lymphoblastoid cell line. Of all siRNAs tested, only the anti-SRSF3 siRNA caused an increase in exon 2 skipping, suggesting that SRSF3 is indeed the key regulator of CD19 splicing. Interestingly, SRSF3 has been shown to interact with PSIP1, a cofactor known to "read" modified histone H3K36me3 (Pradeepa et al., 2012), suggesting a convergence of splicing-based and epigenetics mechanisms. Indeed, exonic regions in genomic DNA are enriched for H3K36me3, and knockdown of Setd2, the H3K36 methyltransferase, results in changes in exon inclusion (Luco et al., 2010; Brown et al., 2012; Hnilicova and Stanek, 2011). Thus, we are currently investigating the connection between the H3K36me3 marks in the CD19 locus and alternative splicing of CD19. Our data could suggest a method of restoring full-length CD19 expression in immunotherapy-resistant cancers using epigenetic drugs. Maude, S L, Noelle, F, Shaw, PA, Aplenc, R, Barrett, DM, Bunin, NJ, Chew, A, Gonzalez, VE, Zheng, Z, Lacey, SF, Mahnke, YD, Melenhorst, JJ, Rheingold, SR, Shen, A, Teachey, DT, Levine, BL, June CH, Porter, DL, and Grupp, SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507-1517. Sotillo, E, Barrett, D, Bagashev, A, Black, K, Lanauze, C, Oldridge, D, Sussman, R, Harrington, C, Chung, EY, Hofmann, TJ, Maude, SL, Martinez, NM, Raman, P, Ruella, M, Allman, D, Jacoby, E, Fry, T, Barash, Y, Lynch, KW, Mackall, C, Maris, J, Grupp, SA, and Thomas-Tikhonenko, A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015AACR Annual Meeting, Philadelphia. Barash Y, Vaquero-Garcia J, González-Vallinas J, Xiong HY, Gao W, Lee LJ, and Frey BJ. AVISPA: a web tool for the prediction and analysis of alternative splicing. Genome Biol 2013; 14(10):R114. Pradeepa, MM, Sutherland, HG, Ule, J, Grimes, GR, and Bickmore, WA. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLOS Genets 2012; 8:e1002717. Luco, RF, Pan, Q, Tominaga, K, Blencowe, BJ, Pereira-Smith, OM, Misteli, T. Regulation of alternative splicing by histone modifications. Science 2010; 327: 996-1000. Brown, SJ, Stoilov, P, and Xing, Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human Mol Genets 2012; 21:R90-R96. Hnilicova, J, and Stanek, D. Where splicing joins chromatin. Nucleus 2011; 2:182-188. Disclosures No relevant conflicts of interest to declare.