After the 3ʹ-end of a eukaryotic pre-mRNA molecule has been cleaved, the RNA polymerase II continues to transcribe, in some cases for hundreds of nucleotides. Once 3ʹ-end cleavage has occurred, the newly synthesized RNA that emerges from the polymerases lacks a 5ʹ cap; this unprotected RNA is rapidly degraded by a 5ʹ →3ʹ exonuclease carried along on the polymerase tail. Apparently, it is this continued RNA degradation that eventually causes the RNA polymerase to release its grip on the template and terminate transcription.
Mature Eukaryotic mRNAs Are Selectively Exported from the Nucleus
Eukaryotic pre-mRNA synthesis and processing take place in an orderly fashion within the cell nucleus. But of the pre-mRNA that is synthesized, only a small fraction—the mature mRNA—is of further use to the cell. Most of the rest—excised introns, broken RNAs, and aberrantly processed pre-mRNAs—is not only useless but potentially dangerous. How does the cell distinguish between the relatively rare mature mRNA molecules it wishes to keep and the overwhelming amount of debris created by RNA processing?
The answer is that, as an RNA molecule is processed, it loses certain proteins and acquires others. For example, we have seen that acquisition of cap-binding complexes, exon junction complexes, and poly-A-binding proteins mark the completion of capping, splicing, and poly-A addition, respectively. A properly completed mRNA molecule is also distinguished by the proteins it lacks. For example, the presence of an snRNP protein would signify incomplete or aberrant splicing. Only when the proteins present on an mRNA molecule collectively signify that processing was successfully completed is the mRNA exported from the nucleus into the cytosol, where it can be translated into protein. Improperly processed mRNAs
Figure 6–35 Some of the major steps in generating the 3ʹ end of a eukaryotic mRNA. This process is much more complicated than the analogous process in bacteria, where the RNA polymerase simply stops at a termination signal and releases both the 3ʹ end of its transcript and the DNA template (see Figure 6–11).
Figure 6–34 Consensus nucleotide sequences that direct cleavage and polyadenylation to form the 3ʹ end of a eukaryotic mRNA. These sequences are encoded in the genome, and specific proteins recognize them—as RNA—after they are transcribed. As shown in Figure 6–35, the hexamer AAUAAA is bound by CPSF and the GU-rich element beyond the cleavage site is bound by CstF; the CA sequence is bound by a third protein factor required for the cleavage step. Like other consensus nucleotide sequences discussed in this chapter (see Figure 6–12), the sequences shown in the figure represent a variety of individual cleavage and polyadenylation signals.
mature 3˜ end of an mRNA molecule and other RNA debris (excised intron sequences, for example) are retained in the nucleus, where they are eventually degraded by the nuclear exosome, a large protein complex whose interior is rich in 3ʹ-to-5ʹ RNA exonucleases (Figure 6–36). Eukaryotic cells thus export only useful RNA molecules to the cytoplasm, while debris is disposed of in the nucleus.
Of all the proteins that assemble on pre-mRNA molecules as they emerge from transcribing RNA polymerases, the most abundant are the hnRNPs (heterogeneous nuclear ribonuclear proteins). Some of these proteins (there are approximately 30 different ones in humans) unwind the hairpin helices in the RNA so that splicing and other signals on the RNA can be read more easily. Others preferentially package the RNA contained in the very long intron sequences typical in complex organisms (see Figure 6–31) and these may play an important role in distinguishing mature mRNA from the debris left over from RNA processing.