Efficient 5′ cap-dependent RNA purification use in identifying and studying subsets of RNA

Abstract

Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in biology. RNA not only carries genetic information in the form of templates and components of the translational machinery for protein synthesis but also directly regulates gene expression as exemplified by micro-RNAs (miRNAs). Recent evidence has demonstrated that 5′ capped and 3′ polyadenylated ends are not restricted to mRNAs, but that they are also present in precursors of both miRNAs and some antisense RNA transcripts. In addition, as many as 40% of transcribed RNAs may lack 3′ poly(A) ends. In concert with the presence of a 5′ cap (m 7 GpppN), the length of the 3′ poly(A) end plays a critical role in determining the translational efficiency, stability, and the cellular distribution of a specific mRNA. RNAs with short or lacking 3′ poly(A) ends, that escape isolation and amplification with oligo(dT)-based methods, provide a challenge in RNA biology and gene expression studies. To circumvent the limitations of 3′ poly(A)-dependent RNA isolation methods, we developed an efficient RNA purification system that binds the 5′ cap of RNA with a high-affinity variant of the cap-binding protein eIF4E. This system can be used in differential selection approaches to isolate subsets of RNAs, including those with short 3′ poly(A) ends that are likely targets of post-transcriptional regulation of gene expression. The length of the 3′ poly(A) ends can be defined using a rapid polymerase chain reaction (PCR)-based approach. © Humana Press.