Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo

Alternative splicing of the bcl-x gene generates two transcripts: the anti-apoptotic bcl-xL isoform and the pro-apoptotic bcl-xS isoform. The ratio between the two isoforms is a key factor in development and in cancer progression. Here, we show that a short antisense chimeric peptide nucleic acid (PNA) oligonucleotide conjugated to a polypeptide containing eight Ser-Arg repeats (SR)(8) can modulate splicing of bcl-x both in vitro and in vivo and induces apoptosis in HeLa cells. The PNA-SR oligo was targeted to a region of bcl-x that does not contain splicing regulatory sequences and was able to override the complex network of splicing enhancers and silencers that regulates the ratio between the two bcl-x isoforms. Thus, PNA-SR oligos are powerful tools that can potentially modulate splice site choice in endogenous genes independent of the presence of other splicing regulatory mechanisms on the target gene

The Negative Regulator of Splicing Element of Rous Sarcoma Virus Promotes Polyadenylation

The Rous sarcoma virus gag gene contains a cis-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3′ long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral env 3′ splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3′ splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3′ LTR and aid in the recruitment of the 3′-end processing machinery

An unexpected ending: Noncanonical 3′ end processing mechanisms

Proper 3′ end processing of a nascent transcript is critical for the functionality of the mature RNA. Although it has long been thought that virtually all long RNA polymerase II transcripts terminate in a poly(A) tail that is generated by endonucleolytic cleavage followed by polyadenylation, noncanonical 3′ end processing mechanisms have recently been identified at several gene loci. Unexpectedly, enzymes with well-characterized roles in other RNA processing events, such as tRNA biogenesis and pre-mRNA splicing, cleave these nascent transcripts to generate their mature 3′ ends despite the presence of nearby polyadenylation signals. In fact, the presence of multiple potential 3′ end cleavage sites is the norm at many human genes, and recent work suggests that the choice among sites is regulated during development and in response to cellular cues. It is, therefore, becoming increasing clear that the selection of a proper 3′ end cleavage site represents an important step in the regulation of gene expression and that the mature 3′ ends of RNA polymerase II transcripts can be generated via multiple mechanisms

Long noncoding RNAs: functional surprises from the RNA world

Most of the eukaryotic genome is transcribed, yielding a complex network of transcripts that includes tens of thousands of long noncoding RNAs with little or no protein-coding capacity. Although the vast majority of long noncoding RNAs have yet to be characterized thoroughly, many of these transcripts are unlikely to represent transcriptional “noise” as a significant number have been shown to exhibit cell type-specific expression, localization to subcellular compartments, and association with human diseases. Here, we highlight recent efforts that have identified a myriad of molecular functions for long noncoding RNAs. In some cases, it appears that simply the act of noncoding RNA transcription is sufficient to positively or negatively affect the expression of nearby genes. However, in many cases, the long noncoding RNAs themselves serve key regulatory roles that were assumed previously to be reserved for proteins, such as regulating the activity or localization of proteins and serving as organizational frameworks of subcellular structures. In addition, many long noncoding RNAs are processed to yield small RNAs or, conversely, modulate how other RNAs are processed. It is thus becoming increasingly clear that long noncoding RNAs can function via numerous paradigms and are key regulatory molecules in the cell

Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence

The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16–25)) in BCa and to consider the opportunity of using circSTAG2(16–25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16–25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16–25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16–25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16–25) activity, we confirmed that circSTAG2(16–25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16–25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16–25) might be considered as a potential biomarker for recurrence prediction