A view of pre-mRNA splicing from RNase R resistant RNAs

Abstract

During pre-mRNA splicing, exons in the primary transcript are precisely connected to generate an mRNA. Intron lariat RNAs are formed as by-products of this process. In addition, some exonic circular RNAs (circRNAs) may also result from exon skipping as by-products. Lariat RNAs and circRNAs are both RNase R resistant RNAs. RNase R is a strong 3' to 5' exoribonuclease, which efficiently degrades linear RNAs, such as mRNAs and rRNAs; therefore, the circular parts of lariat RNAs and the circRNAs can be segregated from eukaryotic total RNAs by their RNase R resistance. Thus, RNase R resistant RNAs could provide unexplored splicing information not available from mRNAs. Analyses of these RNAs identified repeating splicing phenomena, such as re-splicing of mature mRNAs and nested splicing. Moreover, circRNA might function as microRNA sponges. There is an enormous variety of endogenous circRNAs, which are generally synthesized in cells and tissues.

Figure 1

Flow chart of RNase R treatment. Linear RNAs such as mRNAs can be degraded by RNase R treatment. RNase R can also degrade the 3' tail region of an intron lariat RNA. Conversely, the circular part of the lariat RNA and circular RNAs are resistant to RNase R treatment. When total RNA is used as an RNA source, rRNA (major linear RNAs in the total RNA) depletion can help to enrich circular RNAs.

Figure 2

Schematic representation of a model that generates a circRNA. This is one of the most plausible models of circRNA synthesis. The red color indicates head-to-tail splicing. Although this figure is drawn to show that the conventional splicing events in intron 2 to intron 6 happen after the exon skipping event, it is possible that conventional splicing happens before exon skipping. Moreover, even when the lariat RNA contains one single exon, head-to-tail splicing could occur between a downstream donor site and an upstream acceptor site.

Figure 3

Schematic representation of the mature mRNA re-splicing model. Correlated expressions of exon skipped mRNA and exonic lariat RNA (lariat exon) indicated that this splicing model operates for the TSG101 (tumor susceptibility gene 101) and FHIT (fragile histidine triad protein) genes [37]. The first splicing conventionally generates normal mRNA. Although the spliced products are not usually involved in additional splicing processes, normal TSG101 and FHIT mRNAs are probably involved in the second splicing events. Re-splicing of mature mRNA produces multiple exon skipped mRNAs and exonic lariat RNAs.

Figure 4

Schematic representation of exonic lariat RNA and circRNA. The mature mRNA re-splicing produces exonic lariat RNA. Therefore RT-PCR can amplify a product that lacks a potential poly pyrimidine-tract region between the potential branch point and 5' splice site of the re-splicing. The splice site consensus; GU and AG, should be required for the re-splicing. A red asterisk indicates frequent occurrence of the base substitutions [13]. Meanwhile, these features are not applicable in circRNA. Instead, the head-to-tail splicing should precisely connect exons of circRNA. As shown by black asterisk, an exon-exon junction of normal mRNA is used as the splice site in FHIT re-splicing event. However, it is possible that the splice sites of the re-splicing are located in the middle of exons in normal mRNA, such as TSG101 [37].