The expanding transcriptome: the genome as the 'Book of Sand'

Abstract

The central dogma of molecular biology inspired by classical work in prokaryotic organisms accounts for only part of the genetic agenda of complex eukaryotes. First, post-transcriptional events lead to the generation of multiple mRNAs, proteins and functions from a single primary transcript, revealing regulatory networks distinct in mechanism and biological function from those controlling RNA transcription. Second, a variety of populous families of small RNAs (small nuclear RNAs, small nucleolar RNAs, microRNAs, siRNAs and shRNAs) assemble on ribonucleoprotein complexes and regulate virtually all aspects of the gene expression pathway, with profound biological consequences. Third, high-throughput methods of genomic analysis reveal that RNAs other than non-protein-coding RNAs (ncRNAs) represent a major component of the transcriptome that may perform novel functions in gene regulation and beyond. Post-transcriptional regulation, small RNAs and ncRNAs provide an expanding picture of the transcriptome that enriches our views of what genes are, how they operate, evolve and are regulated.

Figure 1

Example of an RNA processing pathway in eukaryotic gene expression. Messenger RNAs are synthesized as precursors containing a 7-methyl-guanidine triphosphate cap at the 5′ end, a polyadenylate tail at the 3′ end and intervening sequences (introns, thin red line) that are eliminated to generate translatable mRNAs where exons (red rectangles) are spliced together. Also represented are some of the components of the spliceosome, the complex that catalyzes intron removal through two consecutive trans-esterification steps. Introns are released in a lariat configuration and are often degraded in the nucleus. Spliceosomal factors include proteins like U2AF and small nuclear RNP particles like U1–U6 snRNPs; many other spliceosomal components are not represented. Most primary transcripts, including rRNAs, tRNAs, snoRNAs and miRNAs, undergo various processing steps catalyzed by protein and/or RNP complexes that eliminate internal or flanking sequences or introduce chemical modifications.

Figure 2

An overview of the eukaryotic transcriptome through examples of its products. Transcription by RNA polymerase I of ribosomal RNA precursors is followed by removal of 5′, 3′ extensions and intervening sequences (blue thin lines) to generate ribosomal RNAs (blue rectangles), which are further modified by pseudouridynilation (Ψ) and ribose methylation (CH3) at specific residues and assemble onto ribosomal subunits (blue circle and ellipse), which are then exported to the cytoplasm, where they mediate protein synthesis. Transcription by RNA polymerase II of mRNA precursors follows the processing pathway depicted in Figure 1. Exonic sequences can also be removed as part of an intron (green rectangle, in the example) to generate alternative mRNAs that can direct the synthesis of distinct protein isoforms. One class of transcripts that do not have extensive coding capacity (ncRNAs) are similarly generated and may play important cellular functions (in the example, by acting as a scaffold for the assembly of functionally connected protein factors, represented as polygons of various shapes). Some introns can generate additional transcripts with important functions in gene regulation. One example are snoRNAs, which assemble onto snoRNP RNP complexes and direct pseudouridynilation and ribose methylation of rRNA and other transcripts. Another example are precursors of miRNAs, which after cleavage by Drosha-type RNases in the nucleus and by Dicer-type enzymes in the cytoplasm, generate 20–28 ds miRNAs that assemble onto RNP complexes that repress translation by binding to 3′ UTRs of mRNAs and/or cause mRNA degradation, depending on the degree of complementarity with their target sequence. Other dsRNAs (e.g. siRNAs) can also trigger mRNA decay through the same mechanism. snoRNAs and miRNA precursors can also be generated from exonic sequences of dedicated transcripts. Small dsRNA fragments generated through bidirectional transcription—shRNAs (often from repetitive DNA—rasiRNAs) and cleavage can also induce transcriptional silencing through histone and DNA methylation.

Figure 3

Transcript diversity and combinatorial regulation of gene expression. Multiple transcripts can be generated from the same gene locus through the use of alternative promoters, alternative polyadenylation sites and alternative splicing. The relative use of these signals is determined by the levels of general factors or by factors specific of particular classes of promoters or splice sites (represented by symbols of various shapes and colors), which may be expressed in a tissue-specific fashion. Coupling between these processes can influence the relative levels of mRNA isoforms. For example, the use of particular promoters or polyadenylation sites can affect the association of splicing regulatory factors and consequently the proportion of alternatively spliced transcripts. Similarly, the combination of different 5′-, 3′-ends and exons can determine the binding of factors and influence the fate of the RNA, including translational efficiency, decay rate or localization. In the example, the length of the 5′ and 3′-most exons allows binding of combinations of factors (e.g. an RNA-binding protein and a miRNA complex) that can modulate the levels of expression of the different protein isoforms.