Proper capping of RNA promotes correct initiation of protein synthesis as well as stability and processing of mRNA in vivo. While an N7-methylguanosine “cap” is present at the 5' end of most naturally occurring eukaryotic mRNAs and many viral RNAs, Anti Reverse Cap Analog (ARCA) is used to cap RNA generated by in vitro transcription (see sidebar, How ARCA Works).

This article describes how Tristan Coady and Dr. Christian Lorson (University of Missouri) use ARCA in their research on spliceosomal uridine-rich, small nuclear RNAs (UsnRNA) and a potential therapy for spinal muscular atrophy patients [1].

Tristan H Coady and Christian L Lorson
Department of Veterinary Pathobiology
Bond Life Sciences Center
University of Missouri
Columbia, MO, 65211

A major emphasis of Dr. Lorson’s research is to understand the molecular genetic mechanism of the neurodegenerative disease, spinal muscular atrophy (SMA) (see sidebar, Spinal Muscular Atrophy & the SMN Genes). The best characterized cellular function of survival motor neuron (SMN), the protein affected in SMA, is the biogenesis of uridine-rich, small nuclear RNAs (UsnRNAs), like U1 snRNA. The UsnRNAs are essential components of the splicesome and are involved in pre-mRNA splicing, so deficiencies in this housekeeping function disrupt protein synthesis.

Recently, Tristan Coady, a graduate student in Dr. Lorson’s group, used an emerging RNA splicing mechanism termed trans-splicing to re-insert exon 7 into transcripts of the SMA modifier gene, SMN2. Trans-splicing conceptually involves an exogenous, trans-splicing RNA (tsRNA) containing an exon that is targeted to a pre-mRNA. The tsRNA is eventually intercalated via splicesome processing into the final mRNA transcript.

The Assay—UsnRNA-Protein Assembly
SMN function can be measured by assaying the ability of cells to assemble the UsnRNA complex (UsnRNP). To perform this assay, U1 snRNA is transcribed in vitro with radiolabeled UTP and a methyl-cap analog. The goal is to create a U1 snRNA that recapitulates endogenously derived RNAs. Cellular lysates are harvested and incubated with the UsnRNA to begin the process of UsnRNP maturation in vitro. The fully assembled UsnRNPs are identified using antibodies specific to the proteins coordinated with the radiolabeled RNA.

Results
Initial examination of UsnRNA-protein assembly revealed rapid degradation of UsnRNAs transcribed with standard methyl-cap analogs; however, when the same process was carried out using the Anti Reverse Cap Analog, the UsnRNAs became more resistant to degradation and yielded a more robust, repeatable signal (Figure 1). Use of ARCA produced a greater number of biologically relevant UsnRNAs due to the orientation of the synthetic methyl cap. Therefore, upon in vitro assembly utilizing tsRNAs targeted to SMN2 transcripts, an increase in SMN protein was found to correlate with overall U1 snRNP assembly (Figure 2).

Figure 1. ARCA-Transcribed U1 snRNAs are More Resistant to Degradation. Wild type HeLa cellular lysate was harvested and incubated with standard (m7G) and ARCA methyl-capped UsnRNAs. Increasing concentrations of lysate are indicated by black triangles. ARCA = Anti Reverse Cap Analog, WT=wild type U1 snRNA, DSm=negative control.

Figure 2. SMA Patient Fibroblasts Transfected with Trans-Splicing RNAs Increase U1 snRNP Assembly in vitro. Fibroblasts were transfected with a plasmid that expresses a trans-splicing RNA for exon 7 of SMN2, and cell lysates were harvested 48 hours later. Increasing amounts of unlabeled U1 snRNA were titrated into reactions to illustrate specificity (black triangles). WT=wild type U1 snRNA, DSm=negative control.

To learn more about this research, see the article in Molecular Therapy [1].

Reference
1. Coady TH, Shababi M,Tullis GE, and Lorson CL (2007) Restoration of SMN function: Delivery of a trans-splicing RNA re-directs SMN2 pre-mRNA splicing. Mol Therapy 15(8): 1471–1478.