As discussed in Chapter 9, analysis of the location of transcribing RNA polymerase II in metazoan cells revealed the surprising result that the polymerase transcribes in the downstream direction, into coding regions, and in the upstream direction, away from coding regions, at nearly equal frequency from most promoters. This finding was confirmed by deep sequencing of small RNAs isolated from metazoan cells, which revealed low levels of short, capped RNAs transcribed from both the sense and antisense strands at CpG island promoters, which account for some 70 percent of mammalian promoters. Indeed, deep sequencing of all cellular RNAs showed that both strands of nearly the entire genome are transcribed, although much of the resulting RNA is present at extremely low concentrations of less than one molecule per cell. This finding raised the question of how the cell deals with such “pervasive transcription.”

Sequence analysis of these low-abundance short, capped RNAs indicates that they are probably prevented from reaching high concentrations by RNA processing and nu clear surveillance for abnormally processed RNAs. Sequencing of RNAs from several cell types has revealed that the antisense RNAs have a higher frequency of AAUAAA poly adenylation signal sequences transcribed from the AT-rich DNA of most metazoans (~60 percent AT in mammals) than do transcripts transcribed in the sense direction into coding regions. Because of the high AT composition of mammalian DNA, an AAUAAA sequence in an antisense transcript is frequently followed by a U-rich sequence that may function as the downstream element of a bona fide pre-mRNA cleavage/ polyadenylation signal (see Figure 1). These cleavage/ polyadenylation signals occur much less frequently in transcripts going into coding regions. Where they do occur in the sequence of pre-mRNAs, in either exons or introns, they usually lie downstream of consensus base-pairing sites for U1 snRNA, which has been found to suppress cleavage/polyadenylation following nearby AAUAAA sequences. This function of U1 snRNA may help to explain why the U1 snRNP is much more abundant than the other spliceosomal snRNPs.

Fig1. Model for cleavage and polyadenylation of pre-mRNAs in mammalian cells. Cleavage and polyadenylation specificity factor (CPSF) binds to the upstream AAUAAA polyadenylation signal. CStF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA; binding of CFI and CFII helps stabilize the complex. Binding of poly(A) polymerase (PAP) then stimulates cleavage at a poly(A) cleavage site, which usually is 15–30 nucleotides 3′ of the upstream polyadenylation signal. The cleavage factors are released, as is the downstream RNA cleavage product, which is rapidly degraded. Bound PAP then adds about 12 A residues at a slow rate to the 3′-hydroxyl group generated by the cleavage reaction. Binding of nuclear poly(A)-binding protein (PABPN1) to the initial short poly(A) tail accelerates the rate of addition by PAP. After 200–250 A residues have been added, PABPN1 signals PAP to stop polymerization.

This is not the case for cleavage/polyadenylation signals used in the processing of 3′ ends of mRNAs because U1 snRNA associates with the 5′ end of the terminal intron, far from the poly(A) site. In addition, as discussed above, transcription by RNA polymerase II usually terminates within ~2 kb following cleavage and polyadenylation of a pre-mRNA. Consequently, the enrichment of poly(A) sites, and the relative lack of binding sites for U1 snRNA, in antisense transcripts may lead to cleavage of most of these transcripts within ~2 kb of the transcription start site by cleavage/polyadenylation factors (see Figure 1), followed by termination of transcription (Figure 2). Cleaved antisense transcripts are probably degraded by the same nuclear exonucleases that degrade introns spliced out of pre-mRNAs and sequences downstream of pre-mRNA cleavage/polyadenylation sites, as well as sequences processed out of rRNA and tRNA pre cursors, discussed in a later section. As a result, even though a large number of polymerases transcribe in the “wrong” direction, most of the transcripts generated in this way are rapidly degraded.

Fig2. RNA transcribed in the "wrong" direction from most promoters in metazoans has a high frequency of polyadenylation signals and a low frequency of binding sites for U1 snRNA. This pattern may account for the termination of transcription in the "wrong" direction after about 2 kb for most of these transcripts. PAS represents polyadenylation signals encoded in the DNA that are transcribed into RNA. Cleavage of transcripts transcribed in the upstream direction (scissors) is proposed to generate free RNA ends that are digested by the nuclear exosome and a nuclear 5′→3′ exonuclease, XRN1. In contrast, pre-mRNAs synthesized by RNA polymerase II transcribing into coding regions have evolved to have few poly adenylation signals. Where they do occur, these signals are usually preceded by a binding site for U1 snRNP, which inhibits cleavage at a nearby PAS (stop sign). However, the PAS used to generate the 3′ end of an mRNA does not have a closely associated U1 RNP binding site. See A. E. Almada et al., 2013, Nature 499:360.

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مواضيع ذات صلة

Micro-RNAs Repress Translation and Induce Degradation of Specific mRNAs

Degradation of mRNAs in the Cytoplasm Occurs by Several Mechanisms

Nuclear Exoribonucleases Degrade RNA That Is Processed Out of Pre-mRNAs

Microchip Electrophoresis

Electrophoresis of RNA

Pulsed-Field Gel Electrophoresis

3′ Cleavage and Polyadenylation of Pre-mRNAs Are Tightly Coupled

Self-Splicing Group II Introns Provide Clues to the Evolution of snRNAs

SR Proteins Contribute to Exon Definition in Long Pre-mRNAs

DNA Sequencing Gels

Nuclear Bodies Are Functionally Specialized Nuclear Domains

Chain Elongation by RNA Polymerase II Is Coupled to the Presence of RNA-Processing Factors