Sharon F. Jamison

Spliceosome-mediated RNA trans -splicing as a tool for gene therapy

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre–trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant β-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

The spliceosome assembly pathway in mammalian extracts.

A mammalian splicing commitment complex was functionally defined by using a template commitment assay. This complex was partially purified and shown to be a required intermediate for complex A formation. The productive formation of this commitment complex required both splice sites and the polypyrimidine tract. U1 small nuclear ribonucleoprotein (snRNP) was the only spliceosomal U snRNP required for this formation. A protein factor, very likely U2AF, is probably involved in the formation of the splicing commitment complex. From the kinetics of appearance of complex A and complex B, it was previously postulated that complex A represents a functional intermediate in spliceosome assembly. Complex A was partially purified and shown to be a required intermediate for complex B (spliceosome) formation. Thus, a spliceosome pathway is for the first time supported by direct biochemical evidence: RNA+U1 snRNP+?U2 auxiliary factor+?Y----CC+U2 snRNP+Z----A+U4/6,5 snRNPs+ beta----B.

Functional Genomics Approach for the Identification of Human Host Factors Supporting Dengue Viral Propagation

Dengue virus (DENV) is endemic throughout tropical regions of the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high-throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle.

HeLa Nuclear Extract: A Modified Protocol

Publisher Summary This chapter focuses on HeLa nuclear extracts. The chapter presents a method that is used for the growth of HeLa cells. HeLa S3 cells (human epithelioid carcinoma, cervix) were obtained from ATCC stocks. The cells are adapted to grow in Jokliks MEM medium; containing 5% defined Bovine Calf Serum, supplemented with penicillin-streptomycin and NaHCO3 at 37°C. The cells were grown in suspension and stirred using magnetic bars at medium speed. All flasks should be tightly sealed in order to keep the carbon dioxide (CO2 ) at appropriate levels. HeLa cells are grown at an ideal density of 5–8 × 105/ml (minimum density should be 3 × 105/ml in 20 l of medium. HeLa cells double approximately every 24 h, and to keep a density of 5–8 × 105/ml, half of the volume of the cell suspension should be discarded daily and the same amount of fresh medium should be added until expansion is required. This chapter also discuses the extract preparation including: materials, methods, and nuclear extraction.Publisher Summary nThis chapter focuses on HeLa nuclear extracts. The chapter presents a method that is used for the growth of HeLa cells. HeLa S3 cells (human epithelioid carcinoma, cervix) were obtained from ATCC stocks. The cells are adapted to grow in Jokliks MEM medium; containing 5% defined Bovine Calf Serum, supplemented with penicillin-streptomycin and NaHCO3 at 37°C. The cells were grown in suspension and stirred using magnetic bars at medium speed. All flasks should be tightly sealed in order to keep the carbon dioxide (CO2 ) at appropriate levels. HeLa cells are grown at an ideal density of 5–8 × 105/ml (minimum density should be 3 × 105/ml in 20 l of medium. HeLa cells double approximately every 24 h, and to keep a density of 5–8 × 105/ml, half of the volume of the cell suspension should be discarded daily and the same amount of fresh medium should be added until expansion is required. This chapter also discuses the extract preparation including: materials, methods, and nuclear extraction.

Erratum: Spliceosome-mediated RNA trans-splicing as a tool for gene therapy (Nature Biotechnology)

Because of a printing error, Figure 1 in Spliceosome-mediated RNA trans-splicing as a tool for gene therapy, by M. Puttaruju, Sharon F. Jamison, Gary Mansfield, Mariano A. Garcia-Blanco, and Lloyd G. Mitchell, wav below:

HeLa Nuclear Extract

Publisher Summary This chapter focuses on HeLa nuclear extracts. The chapter presents a method that is used for the growth of HeLa cells. HeLa S3 cells (human epithelioid carcinoma, cervix) were obtained from ATCC stocks. The cells are adapted to grow in Jokliks MEM medium; containing 5% defined Bovine Calf Serum, supplemented with penicillin-streptomycin and NaHCO3 at 37°C. The cells were grown in suspension and stirred using magnetic bars at medium speed. All flasks should be tightly sealed in order to keep the carbon dioxide (CO2 ) at appropriate levels. HeLa cells are grown at an ideal density of 5–8 × 105/ml (minimum density should be 3 × 105/ml in 20 l of medium. HeLa cells double approximately every 24 h, and to keep a density of 5–8 × 105/ml, half of the volume of the cell suspension should be discarded daily and the same amount of fresh medium should be added until expansion is required. This chapter also discuses the extract preparation including: materials, methods, and nuclear extraction.Publisher Summary nThis chapter focuses on HeLa nuclear extracts. The chapter presents a method that is used for the growth of HeLa cells. HeLa S3 cells (human epithelioid carcinoma, cervix) were obtained from ATCC stocks. The cells are adapted to grow in Jokliks MEM medium; containing 5% defined Bovine Calf Serum, supplemented with penicillin-streptomycin and NaHCO3 at 37°C. The cells were grown in suspension and stirred using magnetic bars at medium speed. All flasks should be tightly sealed in order to keep the carbon dioxide (CO2 ) at appropriate levels. HeLa cells are grown at an ideal density of 5–8 × 105/ml (minimum density should be 3 × 105/ml in 20 l of medium. HeLa cells double approximately every 24 h, and to keep a density of 5–8 × 105/ml, half of the volume of the cell suspension should be discarded daily and the same amount of fresh medium should be added until expansion is required. This chapter also discuses the extract preparation including: materials, methods, and nuclear extraction.