The Signature of the Five-Stranded vRRM Fold Defined by Functional, Structural and Computational Analysis of the hnRNP L Protein.

The RNA recognition motif (RRM) is the far most abundant RNA binding domain. In addition to the typical ß1α1ß2ß3α2ß4 fold, various sub-structural elements have been described and reportedly contribute to the high functional versatility of RRMs. The heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a highly abundant protein of 64 kDa comprising four RRM domains. Involved in many aspects of RNA metabolism, hnRNP L specifically binds to RNAs containing CA repeats or CA-rich clusters. However, a comprehensive structural description of hnRNP L including its sub-structural elements is missing. Here, we present the structural characterization of the RRM domains of hnRNP L and demonstrate their function in repressing exon 4 of SLC2A2. By comparison of the sub-structural elements between the two highly similar paralog families of hnRNP L and PTB, we defined signatures underlying interacting C-terminal coils (ICCs), the RRM34 domain interaction and RRMs with a C-terminal fifth ß-strand, a variation we denoted vRRMs. Furthermore, computational analysis revealed new putative ICC-containing RRM families and allowed us to propose an evolutionary scenario explaining the origins of the ICC and fifth ß-strand sub-structural extensions. Our studies provide insights of domain requirements in alternative splicing mediated by hnRNP L and molecular descriptions for the sub-structural elements. In addition, the analysis presented may help to classify other abundant RRM extensions and to predict structure-function relationships.

SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response.

A redox-regulated SUMO/acetylation switch of HIPK2 controls the survival threshold to oxidative stress.

Moderate concentrations of reactive oxygen species (ROS) serve as coregulatory signaling molecules, whereas exceedingly high concentrations trigger cell death. Here, we identify ROS-induced acetylation of the proapoptotic kinase HIPK2 as a molecular mechanism that controls the threshold discerning sensitivity from resistance toward ROS-mediated cell death. SUMOylation of HIPK2 at permissive ROS concentrations allows the constitutive association of HDAC3 and keeps HIPK2 in the nonacetylated state. Elevated ROS concentrations prevent SUMOylation of HIPK2 and, consequently, reduce association of HDAC3, thus leading to the acetylation of HIPK2. Reconstitution experiments showed that HIPK2-dependent genes cause decreased ROS levels. Although a nonacetylatable HIPK2 mutant enhanced ROS-induced cell death, an acetylation-mimicking variant ensured cell survival even under conditions of high oxidative stress.

Regulation of the tumor suppressor PML by sequential post-translational modifications.

Post-translational modifications (PTMs) regulate multiple biological functions of the promyelocytic leukemia (PML) protein and also the fission, disassembly, and rebuilding of PML nuclear bodies (PML-NBs) during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitutes typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα.

SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo.

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.

Auto- and cross-regulation of the hnRNP L proteins by alternative splicing.

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This "poison exon" is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

Extragenic accumulation of RNA polymerase II enhances transcription by RNA polymerase III.

Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.