Fig 4.

Illustration of how a gene functions by producing different RNAs. Left panel: Flowchart of the routine in studying a gene's function, with emphasis on the ectopic expression approach. Sequencing RT products leads to identification of a gene's mRNA in a cDNA form. Aligning its sequence with gDNA will localize it to a chromosome, which allows us to knock-in or knockout the gene. Continuing to sequence more cDNAs will identify other mRNA variants, which allows us not only to knock down the expression of one, some or all of the variants using such as siRNA but also to ectopically express the mRNAs using cDNAs. For ectopic expression, each cDNA will be cloned into a vector and introduced to cells in culture or in an animal, and the resulting data are used to evaluate the function of this cDNA. Right panel: A gene, which may be expressed in two different cell types (A and B), has two alternative initiation sites and two alternative termination sites for transcription, permitting it to produce four different transcripts. One, some or all four transcripts may have a long 5'-UTR that may harbor multiple uORFs and/or an even-longer 3'-UTR that may contain AltORFs. In one cell type, e.g. normal cells, splicing of one transcript retains all five exons, thus annotated as the wt mRNA, or alternative splicing produces three mRNA variants. In another cell type, e.g. in cancer or another organ or at another developmental stage, the transcripts are spliced to a partly different spectrum of mRNA variants. Some of the mRNAs encode AltORFs as well, resulting in a total of six AltORFs in the two cell types. Moreover, the intron 2 encodes another gene, and its transcripts may be spliced to a wt mRNA with 3 exons (I1, I2 and I3) or, alternatively, to two other mRNA variants in the two cell types. The intron sequences may be processed to different ncRNAs, although only miRNAs and siRNAs are shown for simplicity. More complexly, part of the Crick strand of the DNA may be transcribed to some antisense RNAs as well. Therefore, the global picture about the function of this gene or genomic locus is a collective (but not simply additive) effect of the six mRNA variants and six AltORFs of the parental gene, the three mRNA variants of the nested gene, and all the ncRNAs (miRNAs, siRNAs, piRNAs, snRNAs, exRNAs, circRNAs, and antisense RNAs) in these two cell types. If the parental or the nested gene encodes a transcription factor or a membrane receptor, different heterodimers may be formed among the protein isoforms of the same gene to exert functions as well.