Complex functional coupling exists between transcriptional elongation and pre-mRNA alternative splicing. Pausing sites and changes in the rate of transcription by RNA polymerase II (RNAPII) may therefore have fundamental impacts in the regulation of alternative splicing. Here, we show that the elongation and splicing-related factor TCERG1 regulates alternative splicing of the apoptosis gene Bcl-x in a promoter-dependent manner. TCERG1 promotes the splicing of the short isoform of Bcl-x (Bcl-x s ) through the SB1 regulatory element located in the first half of exon 2. Consistent with these results, we show that TCERG1 associates with the Bcl-x pre-mRNA. A transcription profile analysis revealed that the RNA sequences required for the effect of TCERG1 on Bcl-x alternative splicing coincide with a putative polymerase pause site. Furthermore, TCERG1 modifies the impact of a slow polymerase on Bcl-x alternative splicing. In support of a role for an elongation mechanism in the transcriptional control of Bcl-x alternative splicing, we found that TCERG1 modifies the amount of pre-mRNAs generated at distal regions of the endogenous Bcl-x. Most importantly, TCERG1 affects the rate of RNAPII transcription of endogenous human Bcl-x. We propose that TCERG1 modulates the elongation rate of RNAPII to relieve pausing, thereby activating the proapoptotic Bcl-x S 5= splice site.T he expression of protein-coding genes in eukaryotes is a highly orchestrated process that involves multiple coordinated events. Genomic DNA must be transcribed into precursor mRNAs (pre-mRNA) by RNA polymerase II (RNAPII) and processed through subsequent steps to yield a mature mRNA that is exported from the nucleus to the cytoplasm and used by the translational machinery. The pre-mRNA undergoes several processing steps, including capping, splicing, and cleavage/polyadenylation, which appear to be precisely coordinated with nascent transcript formation (41,44,49). Of these RNA processing mechanisms, alternative splicing occurs as a widespread means to achieve proteomic diversity. Results of deep sequencing-based expression analyses estimate that more than 90% of multiexon human genes undergo alternative splicing (50, 66). The misregulation of alternative splicing underlies multiple diseases, including neurological disorders and cancer (5,19,32,67).Although transcription and alternative splicing can occur independently, both processes are physically and functionally interconnected (44, 49), and this coupling and coordination may be important for the regulation of gene expression. To date, two models have been proposed to explain the link between transcription and splicing. In the recruitment model, the unique carboxylterminal domain (CTD) of RNAPII functions as a "landing pad" for factors involved in pre-mRNA splicing in a manner that is dependent on the phosphorylation of RNAPII and the resulting functional state of the transcriptional complex (4,7,28,38,40,42,43,71). In the kinetic model, an alternative but not exclusive model, the transcript elongation...

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.Splicing and transcriptional elongation are physically and functionally interconnected processes (1, 2). Although both processes can occur autonomously, their coupling and coordination may be important for regulation of gene expression. Coupling of these two processes may influence splicing and alternative splicing regulation. Indeed, promoter composition, transcriptional elongation efficiency, chromatin environment, and recruitment of specific coregulators to the transcriptional complex have been shown to affect alternative splicing decisions in a number of experimental systems (3-6). Yet the molecular mechanisms at work are not understood. The unique carboxyl-terminal domain (CTD) 4 of the large subunit of RNA-PII seems to play a central role in the coupling of splicing, as well as other RNA processing functions, to transcription (7, 8).To provide a framework, we consider two models as follows: the recruiting and kinetic models, which are not mutually exclusive. In the "recruiting model," the CTD functions as a "landing pad" for specific subsets of RNA processing factors in a manner dependent on its phosphorylation pattern and therefore on the functional state of the transcription complex (9). A number of independent research lines have suggested that RNA splicing factors can interact with RNAPII molecules that are hyperphosphorylated on their CTD (10 -14), although these associations might be highly dynamic and transient in vivo. The integrity of the RNAPII CTD has also been shown to influence the recruitment of splicing factors to active transcription sites in the nucleus (15). The "kinetic model" (1) proposes that the rate of elongation of the nascent transcript affects specific alternative splicing decisions by modulating the probability of simultaneous presentation of competing splicing sites. Thus, modulation of transcription elongation efficiency at specific, alternatively spliced regions of genes might constitute a mechanism to regulate splicing decisions (5). Sev...

In recent years the use of solid lipid nanoparticles (SLNs) as transport systems for the delivery of drugs and biomolecules has become particularly important. The use of cationic SLNs developed by the technique of microemulsion, which are complexed with DNA in order to study their application as non-viral vectors in gene therapy, is reported. The nanoparticles are characterized by scanning electron microscopy and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM) and differential scanning calorimetry (DSC). Furthermore, the process of lyophilization of the samples and their stability was studied. The nanoparticles obtained presented a particle size of 340 nm with a positive surface charge of 44 mV and the capability of forming lipoplexes with DNA plasmids was stated.

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