This question concerns the three major RNA modifications discussed in this lesso

Question

This question concerns the three major RNA modifications discussed in this lesson (5' capping, 3' polyadenylation, and splicing)

a. Suppose a pre-mRNA is produced by transcription. Which RNA modification occurs first? Which RNA modification occurs second? Which is the final RNA modification?

b. Ribozymes play an important role in RNA modification. What is a ribozyme? Which RNA modification processes involve ribozymes?

c. What is the function of each of the following molecules in RNA modification? • snRNP • Guanosine nucleoside • CPSF • PolyA polymerase • Guanylyltransferase

d. Introns are often identified by examining the results of hybridization experiments in the electron microscope. If a gene contains two introns, draw an image that represents the result of a hybridization experiment when the template DNA strand is hybridized to the mature mRNA, followed by hybridization with the coding DNA strand.

Explanation / Answer

a) Processing of eukaryotic pre-mRNA occurs in order - 5' Capping --- 3' Polyadenylation ---- Splicing

b) Catalytic RNA or Ribozymes are RNA molecules with catalytic properties – enzymes made of nucleic acid, not protein. Processing of eukaryotic pre-rRNA involves chemical modification, cutting, end trimming and splicing. Splicing of pre-rRNA introns involves ribozymes. Pre-rRNA introns - Group-I and group-II introns are self splicing and therefore act as ribozymes. These self splicing RNA molecules contain active site for intron removal and exon ligation, occurs via two transesterifiaction reactions.

c)  snRNP - These are involved in the splicing of introns fron the pre-mRNA. Introns, short RNA molecules associates with proteins to form small nuclear ribonucleoproteins (snRNPs). These along with proteins attach to the transcript and form a series of complex, spliceosome, within which splicing of introns occur.

Guanosine nucleoside - It is involved int he self splicing reaction of pre-rRNA. Guanosine nucleoside, the exogenous nucleotide attacks at 5' splice spite by its 3' OH group, leading to the addition og Guanine nucleotide or nucleoside in the 5' end of the intron and releases the 5' exon.

Poly (A) polymerase - It is a template independent RNA polymerase that adds adenine residues at an internal site to create a new 3' end to which poly(A) tail is added.

CPSF (CLeavage and polyadenylation specificity) - For polyadenylation in pre-mRNA, a signal is needed for both cleavage and polyadenylation. Polyadenylation is directed by a signal sequence in the mRNA, 5'-AAUAA-3'. This signal sequence is located 10 to 30 nucleotides upstream of the polyadenylation site, present after 5'-CA-3' and followed by GU rich region. Poly A signal sequence and GU rich region is the binding site of CPSF and CstF, respectively.

Guanylyl transferase - It is involved in capping of pre-mRNA. It transfers the GMP moiety from GTP to the 5'-diphosphate of the nascent transcript, creating the guanosine 5'-5'- triphosphate structure. Then methyl transferase transfer methyl group from S-adenosylmethionine to the N-7 position of guanine at the 5' end of the nascent RNA.

d) Introns are identified by R-loop technique. When DNA containing two introns is hybridized with a mature RNA, then two unhybridized segment or loops will observed, This is due to the presence of introns in DNA which will remain unhybridized to the mature mRNA in which the introns are already spliced out.

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