Chronological stages of eukaryotic pre-tRNA processing:

• First, the $5'$ extra leader sequence is cleaved by RNase P endonuclease (C).
• Second, the intron in the anticodon loop is spliced and excised by tRNA endonuclease and ligase (D).
• Third, the $3'$ terminal nucleotides are trimmed by RNase Z and replaced with the CCA trinucleotide by tRNA nucleotidyltransferase (A).
• Fourth, specific internal bases undergo chemical modifications, such as reduction of uridine to dihydrouridine (B).

Structure of the eukaryotic 5' mRNA cap:

• N1 of guanine is not the site of cap methylation.
• N3 is unmethylated in the guanine core.
• The $5'$ cap of eukaryotic mRNA consists of 7-methylguanosine ($\text{m}^7\text{G}$) attached via an unusual $5'\text{-to-}5'$ triphosphate bridge; the guanine base is specifically methylated at the N7 nitrogen atom by mRNA (guanine-N7-)-methyltransferase using SAM as methyl donor.
• N9 is involved in the N-glycosidic bond linking guanine to ribose.

DNA end-labeling enzymatic reactions:

• T4 polynucleotide kinase (PNK) transfers the $\gamma\text{-phosphate}$ from $[\gamma\text{-}^{32}\text{P}]\text{ATP}$ specifically to the free $5'$ hydroxyl end of DNA molecules, achieving $5'$ end-labeling (A).
• Polynucleotide kinase cannot label $3'$ ends.
• Terminal deoxynucleotidyl transferase (TdT) cannot add nucleotides to $5'$ ends.
• Terminal transferase (TdT) adds labeled deoxynucleotides or dideoxynucleotides (e.g., $[\alpha\text{-}^{32}\text{P}]\text{ddATP}$) specifically to the $3'\text{-OH}$ terminus of DNA molecules, achieving $3'$ end-labeling (D).

cDNA synthesis priming requirements:

• Methylated regions do not provide universal primer-binding sites for reverse transcriptase.
• The $5'$ leader sequence is at the opposite end of reverse transcription initiation.
• The $3'\text{-CCA}$ sequence is specific to transfer RNA (tRNA).
• Eukaryotic mature mRNA possesses a polyadenylated tail (poly(A) tail) at its $3'$ terminus; oligo(dT) primers selectively hybridize to this poly(A) tail, allowing reverse transcriptase to synthesize the complementary first-strand cDNA.