RNA processing

01Why RNA is processed

In bacteria, ribosomes start translating an mRNA before its transcription has finished; there is no nucleus between the two. In our cells the RNA polymerases make primary transcripts: precursors that must be changed before they can work. The primary transcripts of mRNAs, pre-mRNAs, are also called heterogeneous nuclear RNA (hnRNA), for their many sizes. The changes happen in the nucleus, mostly while the RNA is still being made, and only a finished mRNA is exported to the cytoplasm.

1. 1

   Capping

   When the RNA is about 25 nucleotides long, a 7-methylguanosine is added to its 5′ end, joined 5′ to 5′ through three phosphates.
2. 2

   Splicing

   Introns are removed and the exons joined, most of them while transcription goes on.
3. 3

   Cleavage and the poly(A) tail

   Just past the signal AAUAAA, the RNA is cut, and poly(A) polymerase adds about 200 adenines to its new 3′ end.
4. 4

   Export

   The finished mRNA, coated with proteins, leaves through a nuclear pore.

02The cap and the tail

The 5′ cap is a guanosine methylated at position 7, joined to the first nucleotide of the RNA the wrong way round, 5′ to 5′, through a triphosphate. Exonucleases that break RNA down from its 5′ end cannot use it, and it is bound by proteins that help export the mRNA and by eIF4E, the part of the initiation complex that binds the cap, so that the small ribosomal subunit is brought to the mRNA (translation).

The poly(A) tail is not copied from the gene. A complex of proteins recognises the AAUAAA signal and a sequence rich in G and U further along, the RNA is cut between them, and poly(A) polymerase adds the adenines. Poly(A)-binding proteins coat the tail, protect the mRNA and help it be translated. In the cytoplasm the tail is slowly shortened, and once it is short the mRNA is broken down, so the tail works as a clock. The mRNAs of the main, replication-dependent histones are the exception: they have no tail and end in a hairpin instead.

03Splicing

Most of a human gene is introns. The protein-coding parts of all our genes together make up only about 1.5 per cent of the genome, while introns are often thousands of nucleotides long and a gene can have dozens of them.

An intron is recognised by three signals: a 5′ splice site beginning with GU, a 3′ splice site ending with AG, and a branch point, an adenine 18 to 40 nucleotides upstream of the 3′ end, followed by a stretch of pyrimidines.

1. 1

   The intron is recognised

   The U1 snRNP pairs with the 5′ splice site and the U2 snRNP with the branch point. The U4, U5 and U6 snRNPs join, and the spliceosome rearranges: U6 replaces U1, and with U2 forms the catalytic core, made of RNA.
2. 2

   The first cut

   The 2′-OH of the branch-point adenine attacks the 5′ splice site. The intron's 5′ end is joined to the branch point, making a loop with a tail, a lariat.
3. 3

   The exons are joined

   The free 3′-OH of the first exon attacks the 3′ splice site. The two exons are joined, and the intron leaves as a lariat and is broken down.

Splicing exchanges one phosphodiester bond for another, so it needs no energy for the joining itself; the spliceosome uses ATP to assemble and rearrange. Some introns in other organisms splice themselves out with no proteins at all, which is one of the ways ribozymes were found.

Alternative splicing

A pre-mRNA can often be spliced in more than one way: an exon may be kept or skipped, or one of two exons used, or a different splice site chosen. About 95 per cent of human genes with more than one exon are spliced in more than one way, which is one reason about 19,500 protein-coding genes give rise to many more proteins. Which version is made depends on the tissue, the stage of development and signals the cell receives.

• In the thyroid, the calcitonin gene gives calcitonin, a hormone that lowers blood calcium; in nerve cells, the same gene, spliced differently, gives calcitonin gene-related peptide, which widens blood vessels.
• A B cell first makes its antibody with a tail that anchors it in the membrane; once activated, it switches to a version without it, which is secreted, by changing where the RNA is cut and spliced.

Checking the mRNA

Splicing leaves a group of proteins, the exon junction complex, on the mRNA at most joints. When a ribosome first reads the mRNA it removes them; if it meets a stop codon while a complex still lies downstream, the stop is premature, and the mRNA is broken down: nonsense-mediated decay. This protects the cell from shortened proteins that could do harm.

04RNA editing

Some RNAs have their sequence changed after they are made, and rarely this changes the protein an mRNA codes for. In the small intestine, an enzyme turns one cytidine of the apolipoprotein B mRNA into uridine, making a CAA codon for glutamine into the stop codon UAA. The liver makes the full protein, apoB-100, for VLDL and LDL; the intestine makes a protein about half as long, apoB-48, for chylomicrons. Other enzymes, ADARs, change adenosines to inosine, which the ribosome reads as guanosine. This is common in our RNA, mostly in repeated sequences outside the parts that code for protein; in a few mRNAs of the brain it changes the protein, for example a receptor for glutamate.

05rRNA and tRNA

Ribosomal RNA is made by RNA polymerase I in the nucleolus as one long precursor (45S), which is cut into the 18S, 5.8S and 28S rRNAs. Before it is cut, small nucleolar RNAs (snoRNAs) guide enzymes to particular nucleotides to add methyl groups or change uridines to pseudouridine. The rRNAs are assembled with ribosomal proteins, brought in from the cytoplasm, into the two subunits, which are exported. The 5S rRNA is made separately by RNA polymerase III.

Transfer RNA is also made as a longer precursor, by RNA polymerase III. Its extra 5′ sequence is cut off by RNase P, an enzyme whose catalytic part is RNA; its extra 3′ end is trimmed; the sequence CCA, to which the amino acid will be attached, is added to the 3′ end; a few tRNAs have a short intron removed; and many of its bases are chemically modified.