Posttranscriptional Modification of  RNA

A primary transcript is the initial, linear, RNA copy of a transcription unit (the segment of DNA between specific initiation and termination sequences). The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. tRNA are further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified both co- and posttranscriptionally.
A. Ribosomal RNA
rRNA of both prokaryotic and eukaryotic cells are generated from long precursor molecules called pre-rRNA. The 23S, 16S, and 5S rRNA of prokaryotes are produced from a single pre-rRNA molecule, as are the 28S, 18S, and 5.8S rRNA of eukaryotes (Fig. 1). [Note: Eukaryotic 5S rRNA is synthesized by RNA pol III and modified separately.] The prerRNA are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further processed (trimmed by exonucleases and modified at some bases and riboses) to produce the required RNA species. [Note: In eukaryotes, rRNA genes are found in long, tandem arrays. rRNA synthesis and processing occur in the nucleolus, with base and sugar modifications facilitated by snoRNA.]

Figure 1: Posttranscriptional processing of eukaryotic ribosomal RNA by ribonucleases (RNases). S = Svedberg unit.
B. Transfer RNA
Both eukaryotic and prokaryotic tRNA are also made from longer precursor molecules that must be modified (Fig. 2). Sequences at both ends of the molecule are removed, and, if present, an intron is removed from the anticodon loop by nucleases. Other posttranscriptional modifications include addition of a –CCA sequence by nucleotidyltransferase to the 3′-terminal end of tRNA and modification of bases at specific positions to produce the unusual bases characteristic of tRNA .

Figure 2: A. Precursor transfer RNA (pre-tRNA) transcript. B. Mature (functional) tRNA after posttranscriptional modification. Modified bases include D (dihydrouracil), ψ (pseudouracil), and m, which means that the base has been methylated.
C. Eukaryotic messenger RNA
The collection of all the primary transcripts synthesized in the nucleus by RNA pol II is known as heterogeneous nuclear RNA (hnRNA). The premRNA components of hnRNA undergo extensive co- and posttranscriptional modification in the nucleus and become mature mRNA.
These modifications usually include the following. [Note: Pol II itself recruits the proteins required for the modifications.]

1. Addition of a 5′-cap: This is the first of the processing reactions for premRNA (Fig. 3). The cap is a 7-methylguanosine attached to the 5′-terminal end of the mRNA through an unusual 5′→5′-triphosphate linkage that is resistant to most nucleases. Creation of the cap requires removal of the γ phosphoryl group from the 5′-triphosphate of the premRNA, followed by addition of guanosine monophosphate (from guanosine triphosphate) by the nuclear enzyme guanylyltransferase.
Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase. S-Adenosylmethionine is the source of the methyl group . Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation .

Figure 3:  Posttranscriptional modification of mRNA showing the 7-methylguanosine cap and polyadenylate (poly-A) tail.
2. Addition of a 3′-poly-A tail: Most eukaryotic mRNA (with several exceptions, including those for the histones) have a chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end (see Fig. 3). This poly-A tail is not transcribed from the DNA but rather is added by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′-end. Tailing terminates eukaryotic transcription. Tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.

3. Splicing: Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns or intervening sequences) that do not code for protein. The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called
splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns (for example, those from histone genes). Others contain a few introns, whereas some, such as the primary transcripts for the α chains of collagen, contain >50 introns that must be removed.
a. Role of small nuclear RNA: In association with multiple proteins, uracil-rich snRNA form five small nuclear ribonucleoprotein particles (snRNP, or “snurp”) designated as U1, U2, U4, U5, and U6 that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Fig.4). [Note: In systemic lupus erythematosus, an autoimmune disease, patients produce antibodies against their own nuclear proteins such as snRNP.]

Figure 4: Splicing. [Note: U1 binds the 5′-donor site, and U2 binds the branch A and the 3′-acceptor site. Addition of U4–U6 completes the complex.] snRNP = small nuclear ribonucleoprotein particle.
b. Mechanism: The binding of snRNP brings the sequences of neighboring exons into the correct alignment for splicing, allowing two transesterification reactions (catalyzed by the RNA of U2, U5, and U6) to occur. The 2′-OH group of an adenine nucleotide (known as the branch site A) in the intron attacks the phosphate at the 5′-end of the intron (splice-donor site), forming an unusual 2′→5′-phosphodiester bond and creating a “lariat” structure (see Fig. 4). The newly freed 3′-OH of exon 1 attacks the 5′-phosphate at the spliceacceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded but may be a precursor for ncRNA such as snoRNA. [Note: The GU and AG sequences at the beginning and end, respectively, of introns are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through pores in the nuclear membrane. [Note: The introns in tRNA  are removed by a different mechanism.]
c. Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that at least 20% of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia, a disease in which the production of the β-globin protein is defective . Splice site mutations can result in exons being skipped (removed) or introns retained. They can also activate cryptic splice sites, which are sites that contain the 5′ or 3′ consensus sequence but are not normally used.
4. Alternative splicing: The pre-mRNA molecules from >90% of human genes can be spliced in alternative ways in different tissues. Because this produces multiple variations of the mRNA and, therefore, of its protein product (Fig. 5), it is a mechanism for producing a large, diverse set of proteins from a limited set of genes. For example, the mRNA for tropomyosin (TM), an actin filament–binding protein of the cytoskeleton (and of the contractile apparatus in muscle cells), undergoes extensive tissue-specific alternative splicing with production of multiple isoforms of the TM protein.

Figure 5:  Alternative splicing patterns in eukaryotic messenger RNA (mRNA). The removal (skipping) of exon 2 from the mRNA in panel B results in a protein product that is different than the one made from the mRNA in panel A.