Splicing

The most common cause of β- Thalassemia is the defect in mRNA splicing of the beta globin gene. Mutations that affect the splicing create aberrant transcripts that are degraded before they are translated. If patients inherit a single mutated gene (thalassemia minor), the disease manifests with a mild anemia. However, patients with homozygous mutations (thalassemia major) have severe transfusion-dependent anemia. Which of the following types of RNA are involved in the splicing process?

A. Ribosomal RNA (r RNA)

B. Small nuclear RNA (sn RNA)

C. Transfer RNA (t RNA)

D. Small cytoplasmic RNA (sc RNA)

E. Heterogeneous nuclear RNA (hn RNA)

The correct answer is- B- Small nuclear RNA (snRNA)

A primary transcript is a linear copy of a transcriptional unit-The segment of DNA between specific initiation and termination sequences. The primary transcript of both prokaryotic and eukaryotic t RNA s and r RNA s are post-transcriptionally modified by removing extra nucleotides. In prokaryotic organisms, the primary transcripts of mRNA-encoding genes begin to serve as translation templates even before their transcription has been completed. This is because the site of transcription is not compartmentalized into a nucleus as it is in eukaryotic organisms. Thus, transcription and translation are coupled in prokaryotic cells. Consequently, prokaryotic mRNAs are subjected to little processing prior to carrying out their intended function in protein synthesis.

Nearly all eukaryotic RNA primary transcripts undergo extensive processing between the time they are synthesized and the time at which they serve their ultimate function, whether it be as mRNA or as a component of the translation machinery such as rRNA, 5S RNA, or tRNA or RNA processing machinery, snRNAs.

Some of the processes involved in the post transcriptional modifications of primary transcript of major RNAs are as follows-

A) Ribosomal RNA

In mammalian cells, the three rRNA molecules (28S, 18S and 5.8S r RNAs) are transcribed as part of a single large precursor molecules called, Pre ribosomal RNAs. The pre ribosomal RNAs are cleaved by ribonucleases to yield intermediate-sized pieces of r RNAs, which are further trimmed to produce the required r RNA species. Eukaryotic 5S rRNA is synthesized by RNA polymerase III and modified separately.

B) Transfer RNA

The tRNA molecules serve as adapter molecules for the translation of mRNA into protein sequences. Both eukaryotic and prokaryotic transfer RNA s are made from longer precursor molecules that must be modified. The basic mechanisms involved are:

1) Splicing- An intron must be removed from the anticodon loop (Figure-1)

2) Trimming- The sequences at both the 5′ and 3′ ends of the molecule are trimmed (Figure-1).

3) Base modifications

The tRNAs contain many modifications of the standard bases A, U, G, and C, including methylation, reduction, deamination, and rearranged glycosidic bonds. Further modification of the tRNA molecules includes nucleotide alkylations,

4) CCA attachment (Figure-1)

The attachment of the characteristic CpCpAOH terminal at the 3′ end of the molecule by the enzyme nucleotidyl transferase is the most important modification.

Modifications of t RNA

Figure-1-The extra nucleotides at both 5′ and 3′ ends of t RNA are removed, an intron from the anticodon arm is removed, bases are modified (not shown here) and CCA arm is attached to form the mature functional t RNA.s

C) Eukaryotic mRNA

The primary transcripts are extensively modified in the nucleus after transcription. These modifications include-

a) 5′ Capping

Mammalian mRNA molecules contain a 7-methylguanosine tri phosphate cap structure at their 5′ terminal. The 5 cap of the RNA transcript is required both for efficient translation initiation and protection of the 5′ end of mRNA from attack by 5-‘3’ exonucleases.

b) Addition of poly A tail

Poly (A) tail is added to the 3′ end of mRNA molecule in a post transcriptional processing step. The poly (A) tail appears to protect the 3′ end of mRNA from 3′- 5′ exonuclease attack. Histone and interferon’s mRNAs lack poly A tail.

c) Removal of introns (Splicing)

Intons or intervening sequences are the RNA sequences which do not code for the proteins. These introns are removed from the primary transcript in the nucleus, exons (coding sequences) are ligated to form the mRNA molecule, and the mRNA molecule is transported to the cytoplasm.

The steps of splicing are as follows-

i) Role of small nuclear RNA (sn RNA) and Spliceosome

The molecular machine that accomplishes the task of splicing is known as the spliceosome. Spliceosome consist of the primary transcript, five small nuclear RNAs (U1, U2, U5, U4, and U6) and more than 60 proteins. Collectively, these form a small ribonucleoprotein (snRNP) complex, sometimes called a “snurp” (snRNPs) (Figure-2).Snurps are thought to position the RNA segments for the necessary splicing reactions. These facilitate the splicing of exon segments by forming base pairs with the consensus sequence at each end of the intron.

Spliceosome

Figure-2 Spliceosome assembly at the splice site. The binding of snRNPs brings the sequences of the neighboring exons in to the correct alignment for splicing.

Clinical significance

1) Antibodies against snRNPs

In systemic Lupus Erythematosus (SLE), an auto immune disease, the antibodies are produced against host proteins, including snRNPs.

2) Mutations at the splice site

Mutations at the splice site can lead to improper splicing and the production of aberrant proteins . For example some cases of Beta thalassemia (as mentioned in the question) are as a result of incorrect splicing of beta globin mRNA due to mutation at the splice site.

Biological significance

Alternative Splicing

Alternative patterns of RNA splicing are adapted for the synthesis of tissue-specific proteins. The pre-m RNA molecules from some genes can be spliced in two or more alternative ways in different tissues. This produces multiple variations of the m RNA and thus diverse set of proteins can be synthesized from a given set of genes.

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