Splice switching an oncogenic ratio of SmgGDS isoforms as a strategy to diminish malignancy.

The chaperone protein SmgGDS promotes cell-cycle progression and tumorigenesis in human breast and nonsmall cell lung cancer. Splice variants of SmgGDS, named SmgGDS-607 and SmgGDS-558, facilitate the activation of oncogenic members of the Ras and Rho families of small GTPases through membrane trafficking via regulation of the prenylation pathway. SmgGDS-607 interacts with newly synthesized preprenylated small GTPases, while SmgGDS-558 interacts with prenylated small GTPases. We determined that cancer cells have a high ratio of SmgGDS-607:SmgGDS-558 (607:558 ratio), and this elevated ratio is associated with reduced survival of breast cancer patients. These discoveries suggest that targeting SmgGDS splicing to lower the 607:558 ratio may be an effective strategy to inhibit the malignant phenotype generated by small GTPases. Here we report the development of a splice-switching oligonucleotide, named SSO Ex5, that lowers the 607:558 ratio by altering exon 5 inclusion in SmgGDS pre-mRNA (messenger RNA). Our results indicate that SSO Ex5 suppresses the prenylation of multiple small GTPases in the Ras, Rho, and Rab families and inhibits ERK activity, resulting in endoplasmic reticulum (ER) stress, the unfolded protein response, and ultimately apoptotic cell death in breast and lung cancer cell lines. Furthermore, intraperitoneal (i.p.) delivery of SSO Ex5 in MMTV-PyMT mice redirects SmgGDS splicing in the mammary gland and slows tumorigenesis in this aggressive model of breast cancer. Taken together, our results suggest that the high 607:558 ratio is required for optimal small GTPase prenylation, and validate this innovative approach of targeting SmgGDS splicing to diminish malignancy in breast and lung cancer.

Evidence that a threshold of serine/arginine-rich (SR) proteins recruits CFIm to promote rous sarcoma virus mRNA 3' end formation.

The weak polyadenylation site (PAS) of Rous sarcoma virus (RSV) is activated by the juxtaposition of SR protein binding sites within the spatially separate negative regulator of splicing (NRS) element and the env RNA splicing enhancer (Env enhancer), which are far upstream of the PAS. Juxtaposition occurs by formation of the NRS - 3' ss splicing regulatory complex and is thought to provide a threshold of SR proteins that facilitate long-range stimulation of the PAS. We provide evidence for the threshold model by showing that greater than three synthetic SR protein binding sites are needed to substitute for the Env enhancer, that either the NRS or Env enhancer alone promotes polyadenylation when the distance to the PAS is decreased, and that SR protein binding sites promote binding of the polyadenylation factor cleavage factor I (CFIm) to the weak PAS. These observations may be relevant for cellular PASs.

A novel otoferlin splice-site mutation in siblings with auditory neuropathy spectrum disorder.

We characterize a novel otoferlin mutation discovered in a sibling pair diagnosed with auditory neuropathy spectrum disorder and investigate auditory nerve function through their cochlear implants. Genetic sequencing revealed a homozygous mutation at the otoferlin splice donor site of exon 28 (IVS28 + 1G>T) in both siblings. Functional investigation showed that the intronic sequence between exons 28 and 29 was retained in the mutated minigenes that were expressed in 293T cells. Auditory nerve compound action potential recovery functions in the siblings demonstrated different rates of neural recovery, with sibling AN1 showing rapid recovery (1.14 ms) and AN2 showing average recovery (0.78 ms) compared to subjects with sensorineural hearing loss (average: adults 0.71 ms, children 0.85 ms). Differences in neural recovery were consistent with speech perception differences between the siblings. Genotype information may indicate site of lesion in hearing loss; however, additional, as yet, unknown factors may impact clinical outcomes and must be considered.

Juxtaposition of two distant, serine-arginine-rich protein-binding elements is required for optimal polyadenylation in Rous sarcoma virus.

The Rous sarcoma virus (RSV) polyadenylation site (PAS) is very poorly used in vitro due to suboptimal upstream and downstream elements, and yet ∼85% of viral transcripts are polyadenylated in vivo. The mechanisms that orchestrate polyadenylation at the weak PAS are not completely understood. It was previously shown that serine-arginine (SR)-rich proteins stimulate RSV PAS use in vitro and in vivo. It has been proposed that viral RNA polyadenylation is stimulated through a nonproductive splice complex that forms between a pseudo 5' splice site (5'ss) within the negative regulator of splicing (NRS) and a downstream 3'ss, which repositions NRS-bound SR proteins closer to the viral PAS. This repositioning is thought to be important for long-distance poly(A) stimulation by the NRS. We report here that a 308-nucleotide deletion downstream of the env 3'ss decreased polyadenylation efficiency, suggesting the presence of an additional element required for optimal RSV polyadenylation. Mapping studies localized the poly(A) stimulating element to a region coincident with the Env splicing enhancer, which binds SR proteins, and inactivation of the enhancer and SR protein binding decreased polyadenylation efficiency. The positive effect of the Env enhancer on polyadenylation could be uncoupled from its role in splicing. As with the NRS, the Env enhancer also stimulated use of the viral PAS in vitro. These results suggest that a critical threshold of SR proteins, achieved by juxtaposition of SR protein binding sites within the NRS and Env enhancer, is required for long-range polyadenylation stimulation.

A glycine-rich domain of hnRNP H/F promotes nucleocytoplasmic shuttling and nuclear import through an interaction with transportin 1.

Heterogeneous nuclear ribonucleoprotein (hnRNP) H and F are members of a closely related subfamily of hnRNP proteins that are implicated in many aspects of RNA processing. hnRNP H and F are alternative splicing factors for numerous U2- and U12-dependent introns. The proteins have three RNA binding domains and two glycine-rich domains and localize to both the nucleus and cytoplasm, but little is known about which domains govern subcellular localization or splicing activity. We show here that the central glycine-tyrosine-arginine-rich (GYR) domain is responsible for nuclear localization, and a nonclassical nuclear localization signal (NLS) was mapped to a short, highly conserved sequence whose activity was compromised by point mutations. Glutathione S-transferase (GST) pulldown assays demonstrated that the hnRNP H NLS interacts with the import receptor transportin 1. Finally, we show that hnRNP H/F are transcription-dependent shuttling proteins. Collectively, the results suggest that hnRNP H and F are GYR domain-dependent shuttling proteins whose posttranslational modifications may alter nuclear localization and hence function.

Articular cartilage vesicles contain RNA.

Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 microg RNA/80 microg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.

RNA processing control in avian retroviruses.

Upon integration into the host chromosome, retroviral gene expression requires transcription by the host RNA polymerase II, and viral messages are subject RNA processing events including 5'-end capping, pre-mRNA splicing, and polyadenylation. At a minimum, RNA splicing is required to generate the env mRNA, but viral replication requires substantial amounts of unspliced RNA to serve as mRNA and for incorporation into progeny virions as genomic RNA. Therefore, splicing has to be controlled to preserve the large unspliced RNA pool. Considering the current view that splicing and polyadenylation are coupled, the question arises as to how genome-length viral RNA is efficiently polyadenylated in the absence of splicing. Polyadenylation of many retroviral mRNAs is inefficient; in avian retroviruses, approximately 15 percent of viral transcripts extend into and are polyadenylated at downstream host genes, which often has profound biological consequences. Retroviruses have served as important models to study RNA processing and this review summarizes a body of work using avian retroviruses that has led to the discovery of novel RNA splicing and polyadenylation control mechanisms.

Characterization of Rous sarcoma virus polyadenylation site use in vitro.

Polyadenylation of Rous sarcoma virus (RSV) RNA is inefficient, as approximately 15% of RSV RNAs represent read-through transcripts that use a downstream cellular polyadenylation site (poly(A) site). Read-through transcription has implications for the virus and the host since it is associated with oncogene capture and tumor induction. To explore the basis of inefficient RSV RNA 3'-end formation, we characterized RSV polyadenylation in vitro using HeLa cell nuclear extracts and HEK293 whole cell extracts. RSV polyadenylation substrates composed of the natural 3' end of viral RNA and various lengths of upstream sequence showed little or no polyadenylation, indicating that the RSV poly(A) site is suboptimal. Efficiently used poly(A) sites often have identifiable upstream and downstream elements (USEs and DSEs) in close proximity to the conserved AAUAAA signal. The sequences upstream and downstream of the RSV poly(A) site deviate from those found in efficiently used poly(A) sites, which may explain inefficient RSV polyadenylation. To assess the quality of the RSV USEs and DSEs, the well-characterized SV40 late USEs and/or DSEs were substituted for the RSV elements and vice versa, which showed that the USEs and DSEs from RSV are suboptimal but functional. CstF interacted poorly with the RSV polyadenylation substrate, and the inactivity of the RSV poly(A) site was at least in part due to poor CstF binding since tethering CstF to the RSV substrate activated polyadenylation. Our data are consistent with poor polyadenylation factor binding sites in both the USE and DSE as the basis for inefficient use of the RSV poly(A) site and point to the importance of additional elements within RSV RNA in promoting 3' end formation.

Serine/arginine-rich proteins contribute to negative regulator of splicing element-stimulated polyadenylation in rous sarcoma virus.

Rous sarcoma virus (RSV) requires large amounts of unspliced RNA for replication. Splicing and polyadenylation are coupled in the cells they infect, which raises the question of how viral RNA is efficiently polyadenylated in the absence of splicing. Optimal RSV polyadenylation requires a far-upstream splicing control element, the negative regulator of splicing (NRS), that binds SR proteins and U1/U11 snRNPs and functions as a pseudo-5' splice site that interacts with and sequesters 3' splice sites. We investigated a link between NRS-mediated splicing inhibition and efficient polyadenylation. In vitro, the NRS alone activated a model RSV polyadenylation substrate, and while the effect did not require the snRNP-binding sites or a downstream 3' splice site, SR proteins were sufficient to stimulate polyadenylation. Consistent with this, SELEX-binding sites for the SR proteins ASF/SF2, 9G8, and SRp20 were able to stimulate polyadenylation when placed upstream of the RSV poly(A) site. In vivo, however, the SELEX sites improved polyadenylation in proviral clones only when the NRS-3' splice site complex could form. Deletions that positioned the SR protein-binding sites closer to the poly(A) site eliminated the requirement for the NRS-3' splice site interaction. This indicates a novel role for SR proteins in promoting RSV polyadenylation in the context of the NRS-3' splice site complex, which is thought to bridge the long distance between the NRS and poly(A) site. The results further suggest a more general role for SR proteins in polyadenylation of cellular mRNAs.

Urea-nuclease treatment of concentrated retrovirions preserves viral RNA and removes polymerase chain reaction-amplifiable cellular RNA and DNA.

Cellular nucleic acids can interfere with the molecular cloning of retroviruses, a problem that is particularly serious with viruses propagated in lymphoblastoid cells that release large amounts of microvesicles and other cellular components. The approach taken to circumvent such problems involved first suspending viral pellets in water to allow any residual microvesicles to swell and perhaps lyse during overnight or longer incubation periods. Urea was then added to a concentration of 1.5-2.0 M to uncoil proteins that may protect nucleic acids from hydrolysis on the further addition of Micrococcal nuclease and ribonuclease A, both of which remain enzymatically active in molar urea solutions. The viral RNA was extracted and residual DNA removed by deoxyribonuclease I treatments. The utility of the method was demonstrated with two different retroviruses, a Moloney murine leukemia virus variant and Rous sarcoma virus, such that viral RNA thus purified was shown to be free of contamination by PCR-amplifiable cellular GAPDH mRNA and ribosomal RNA. This general approach should be applicable to viruses of any type in circumstances where contamination by cellular RNA and DNA poses a problem.

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. METHODS: We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G). RESULTS: Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. CONCLUSION: This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

The retrovirus RNA trafficking granule: from birth to maturity.

Post-transcriptional events in the life of an RNA including RNA processing, transport, translation and metabolism are characterized by the regulated assembly of multiple ribonucleoprotein (RNP) complexes. At each of these steps, there is the engagement and disengagement of RNA-binding proteins until the RNA reaches its final destination. For retroviral genomic RNA, the final destination is the capsid. Numerous studies have provided crucial information about these processes and serve as the basis for studies on the intracellular fate of retroviral RNA. Retroviral RNAs are like cellular mRNAs but their processing is more tightly regulated by multiple cis-acting sequences and the activities of many trans-acting proteins. This review describes the viral and cellular partners that retroviral RNA encounters during its maturation that begins in the nucleus, focusing on important events including splicing, 3' end-processing, RNA trafficking from the nucleus to the cytoplasm and finally, mechanisms that lead to its compartmentalization into progeny virions.

Heterogeneous nuclear ribonucleoprotein H is required for optimal U11 small nuclear ribonucleoprotein binding to a retroviral RNA-processing control element: implications for U12-dependent RNA splicing.

An RNA-processing element from Rous sarcoma virus, the negative regulator of splicing (NRS), represses splicing to generate unspliced RNA that serves as mRNA and as genomic RNA for progeny virions and also promotes polyadenylation of the unspliced RNA. Integral to NRS function is the binding of U1 small nuclear ribonucleoprotein (snRNP), but its binding is controlled by U11 snRNP that binds to an overlapping site. U11 snRNP, the U1 counterpart for splicing of U12-dependent introns, binds the NRS remarkably well and requires G-rich elements just downstream of the consensus U11 binding site. We present evidence that heterogeneous nuclear ribonucleoprotein (hnRNP) H binds to the NRS G-rich elements and that hnRNP H is required for optimal U11 binding in vitro. It is further shown that hnRNP H (but not hnRNP F) can promote U11 binding and splicing from the NRS in vivo when tethered to the RNA as an MS2 fusion protein. Interestingly, 17% of the naturally occurring U12-dependent introns have at least two potential hnRNP H binding sites positioned similarly to the NRS. For two such introns from the SCN4A and P120 genes, we show that hnRNP H binds to each in a G-tract-dependent manner, that G-tract mutations strongly reduce splicing of minigene RNA, and that tethered hnRNP H restores splicing to mutant RNA. In support of a role for hnRNP H in both splicing pathways, hnRNP H antibodies co-precipitate U1 and U11 small nuclear ribonucleoproteins. These results indicate that hnRNP H is an auxiliary factor for U11 binding to the NRS and that, more generally, hnRNP H is a splicing factor for a subset of U12-dependent introns that harbor G-rich elements.

Two regions promote U11 small nuclear ribonucleoprotein particle binding to a retroviral splicing inhibitor element (negative regulator of splicing).

The Rous sarcoma virus (RSV) negative regulator of splicing (NRS) is an RNA element that represses splicing and promotes polyadenylation of viral RNA. The NRS acts as a pseudo 5' splice site (ss), and serine-arginine (SR) proteins, U1snRNP, and U6 small nuclear ribonucleoproteins (snRNPs) are implicated in its function. The NRS also efficiently binds U11 snRNP of the U12-dependent splicing pathway, which is interesting, because U11 binds only poorly to authentic substrates that lack a U12-type 3' splice site. It is of considerable interest to understand how the low abundance U11 snRNP binds the NRS so well. Here we show that U11 can bind the NRS as a mono-snRNP in vitro and that a G-rich element located downstream of the U11 site is required for efficient binding. Mutational analyses indicated that two of four G tracts in this region were important for optimal U11 binding and that the G-rich region did not function indirectly by promoting U1 snRNP binding to an overlapping site. Surprisingly, inactivation of U2 snRNP also decreased U11 binding to the NRS. The NRS harbors a branch point-like/pyrimidine tract sequence (BP/Py) just upstream of the U1/U11 site that is characteristic of 3' splice sites. Deletion of this region decreased U2 and U11 binding, and deletion of the G-rich region also reduced U2 binding. The G element, but not the BP/Py sequence, was also required for U11 binding to the NRS in vivo as assessed by minor class splicing from the NRS to a minor class 3'ss from the P120 gene. These results indicate that efficient U11 binding to the isolated NRS involves at least two elements in addition to the U11 consensus sequence and may have implications for U11 binding to authentic splicing substrates.

Position dependence of the Rous sarcoma virus negative regulator of splicing element reflects proximity to a 5' splice site.

Rous sarcoma virus (RSV) requires incomplete splicing of its viral transcripts to maintain efficient replication. A splicing inhibitor element, the negative regulator of splicing (NRS), is located near the 5' end of the RNA but the significance of this positioning is not known. In a heterologous intron the NRS functions optimally when positioned close to the authentic 5' splice site. This observation led us to investigate the basis of the position dependence. Four explanations were put forth and stressed the role of three major elements involved in splicing, the 3' splice site, the 5' splice site, and the 5' end cap structure. NRS function was unrelated to its position relative to the 3' splice site or the cap structure and appeared to depend on its position relative to the authentic 5' splice site. We conclude that position dependence may reflect distance constraints necessary for competition of the NRS with the authentic 5' splice site for pairing with the 3' splice sites.

mRNA decay: x (XRN1) marks the spot.

Degradation of mRNA is a vital aspect of gene expression. In yeast, Dcp1p, Dcp2p, Lsm1-7p, and Xrn1p are required for mRNA decay and are localized within discrete cytoplasmic foci; in the May 2 issue of Science, Sheth and Parker provide compelling evidence that these foci represent sites for mRNA decay.

Efficient polyadenylation of Rous sarcoma virus RNA requires the negative regulator of splicing element.

Rous sarcoma virus pre-mRNA contains an element known as the negative regulator of splicing (NRS) that acts to inhibit viral RNA splicing. The NRS binds serine/arginine-rich (SR) proteins, hnRNP H and the U1/U11 snRNPs, and appears to inhibit splicing by acting as a decoy 5' splice site. Deletions within the gag gene that encompass the NRS also lead to increased read-through past the viral polyadenylation site, suggesting a role for the NRS in promoting polyadenylation. Using NRS-specific deletions and mutations, we show here that a polyadenylation stimulatory activity maps directly to the NRS and is most likely dependent upon SR proteins and U1 and/or U11 snRNP. hnRNP H does not appear to mediate splicing control or stimulate RSV polyadenylation, since viral RNAs containing hnRNP H-specific mutations were spliced and polyadenylated normally. However, the ability of hnRNP H mutations to suppress the read-through caused by an SR protein mutation suggests the potential for hnRNP H to antagonize polyadenylation. Interestingly, disruption of splicing control closely correlated with increased read-through, indicating that a functional NRS is necessary for efficient RSV polyadenylation rather than binding of an individual factor. We propose a model in which the NRS serves to enhance polyadenylation of RSV unspliced RNA in a process analogous to the stimulation of cellular pre-mRNA polyadenylation by splicing complexes.