Biogenesis of miRNA: Canonical and Non-Canonical Pathways

RNA molecules are categorized into three main types based on their functions in protein synthesis: messenger RNA (mRNA) serves as the only coding RNA that carries genetic information from DNA to ribosomes for protein translation; transfer RNA (tRNA) acts as an adapter molecule that recognizes mRNA codons through its anticodon loop and delivers specific amino acids to the growing polypeptide chain; and ribosomal RNA (rRNA) forms the structural and catalytic core of ribosomes, with prokaryotes containing 5S, 16S, and 23S rRNA in their ribosomes while eukaryotes contain 5S, 5.8S, 18S, and 28S rRNA. Beyond these three types, RNA can be classified into coding RNA (only mRNA) and non-coding RNA, which includes housekeeping RNAs essential for translation (tRNA and rRNA) and regulatory RNAs (such as lncRNAs, miRNAs, siRNAs, snoRNAs, snRNAs, and piRNAs) that modulate gene expression and cellular processes.

RNA differs from DNA as a single-stranded polynucleotide with ribose sugar and uracil base. In eukaryotic cells, RNA distribution is approximately 50% in ribosomes/ER, 25% in cytoplasm, 15% in mitochondria, and 10% in nucleus/nucleolus. RNA classifies into coding (mRNA) and non-coding categories. Non-coding RNA further divides into housekeeping (tRNA, rRNA) and regulatory types (long non-coding RNA >200 nt, small non-coding RNA <200 nt). Small non-coding RNA includes miRNA, snoRNA, siRNA, snRNA, and piRNA, each serving specialized regulatory roles in gene expression.

RNA is classified into coding RNA (mRNA) and non-coding RNA (ncRNA). mRNA carries genetic information for protein synthesis and features a characteristic structure: a 5' guanine phosphate cap protecting against degradation and facilitating ribosome recognition, a central coding region containing the genetic message, and a 3' poly-A tail of 50-250 adenines enhancing stability and translation efficiency. Untranslated regions (UTRs) between these components contain regulatory elements controlling gene expression. Non-coding RNA encompasses housekeeping types (tRNA, rRNA) involved in structural functions and regulatory types including piRNAs (DNA integrity), miRNAs (gene regulation), snoRNAs (processing), siRNAs (genetic engineering), and snRNAs (pre-mRNA splicing).

RNA (ribonucleic acid) is classified into coding and non-coding types. Coding RNAs include messenger RNA (mRNA), which carries genetic information from DNA to ribosomes for protein synthesis. Non-coding RNAs include ribosomal RNA (rRNA, 60-80% of total cellular RNA), transfer RNA (tRNA, 15-30%), long non-coding RNAs, small nuclear RNAs (snRNAs) involved in splicing, and microRNAs (miRNAs) and small interfering RNAs (siRNAs) involved in gene regulation and gene silencing. RNA molecules are single-stranded polymers of ribonucleotides and can exhibit catalytic activity (ribozymes).

RNA molecules are broadly classified into coding and non-coding categories based on whether they are translated into proteins. Coding RNA exclusively includes messenger RNA (mRNA), which carries genetic information from DNA to ribosomes for protein synthesis. Non-coding RNA encompasses housekeeping RNAs essential for cellular maintenance (tRNAs and rRNAs) and regulatory RNAs that control gene expression. Regulatory RNAs are further divided by size: long non-coding RNAs (>200 nucleotides) and small non-coding RNAs (<200 nucleotides), including microRNAs (~20 nucleotides) that regulate gene expression post-transcriptionally.

In eukaryotic gene regulation, promoters are DNA sequences where RNA polymerase binds to initiate transcription, while transcription factors are proteins that bind to specific DNA sequences to regulate gene expression; enhancers are regulatory DNA elements that can increase transcription rates when bound by activator proteins, and these components work together with the transcription initiation complex to control when and how much a gene is expressed.

Gene expression in eukaryotes operates at five levels: DNA, transcriptional, post-transcriptional, translational, and post-translational. Housekeeping genes are constitutively expressed, while regulated genes respond to stimuli. The central dogma describes DNA→RNA→protein flow, with eukaryotes having additional post-transcriptional processing. DNA-level regulation includes epigenetic modifications (acetylation/methylation of histones controlling gene accessibility), gene amplification (increasing gene copies, e.g., dihydrofolate reductase in Methotrexate resistance), and gene rearrangement (recombining gene segments, e.g., immunoglobulin diversity). Transcription initiation requires ordered assembly of transcription factors at promoter regions containing TATA boxes, with TBP recruiting TFII-D, TFII-A, TFII-B, TFII-F, TFII-E, and RNA polymerase II. Transcription factors recognize DNA through supersecondary structure motifs including zinc finger, leucine zipper, and helix-turn-helix motifs.

Gene expression is the process by which genetic information creates functional products. Genes are classified as constitutive (expressed in all cells, like ATP synthase) or regulated (expressed under specific conditions, like insulin). The video uses a music analogy: constitutive genes are like drums maintaining constant rhythm, while regulated genes are like triangles adding accents at specific moments. Eukaryotic transcription requires three components: RNA polymerase, basic transcription factors (which help RNA polymerase bind to promoters), and transcription factors (activators and repressors). Regulatory regions include enhancers (increase transcription) and silencers (decrease transcription). Eukaryotic DNA contains promoters, regulatory regions, structural genes, and regulatory genes, enabling sophisticated multi-layered control of gene expression.

Gene expression follows the central dogma: DNA → RNA → protein. Cells require varying amounts of proteins based on developmental, growth, and physiological demands, necessitating precise regulation of transcription and translation. Regulatory mechanisms include trans-regulatory mechanisms (protein-based, transcription factor binding) and cis-regulatory mechanisms (DNA elements near or far from genes). Promoters are short DNA sequences where RNA polymerase binds to initiate transcription, requiring minimal promoter elements. Enhancers are regulatory sequences located 3-4 kilobase pairs away from genes that can still regulate transcription by interacting with transcription factors and cofactors to modulate transcription frequency and speed.

In eukaryotes, RNA processing involves three major steps: 5' capping (adding a methylated guanine cap to protect mRNA and facilitate nuclear export), 3' polyadenylation (adding a poly-A tail to increase mRNA stability and aid in translation), and splicing (removing introns and joining exons via the spliceosome complex containing U1, U2, U4, U5, and U6 snRNPs); these processes convert pre-mRNA into mature mRNA that can be transported to the cytosol for protein synthesis.

Eukaryotic pre-mRNA undergoes three major processing steps before becoming mature mRNA: (1) Capping - addition of a methylated guanosine triphosphate to the 5' end; (2) Polyadenylation - addition of 200-300 adenine nucleotides to the 3' end; (3) Splicing - removal of introns (non-coding regions) and joining of exons (coding regions) by the spliceosome (composed of snRNAs and proteins). This processing allows for alternative splicing, where different combinations of exons can be joined to produce different proteins from the same gene.

Eukaryotic mRNA undergoes three major processing steps: (1) Capping begins as soon as 30 nucleotides are synthesized, (2) Splicing removes introns and joins exons, (3) Polyadenylation adds a poly-A tail to the 3' end of the last exon. RNA polymerase II synthesizes mRNA, while RNA polymerase I and III synthesize rRNA and tRNA respectively. All three polymerases are essential for translation and cellular function.

In eukaryotic cells, pre-mRNA undergoes three essential processing steps before becoming mature mRNA: (1) Capping at the 5' end involves removing the terminal phosphate and attaching a methylated guanosine cap, which protects the mRNA and facilitates nuclear export; (2) Splicing removes non-coding introns and joins coding exons together using the spliceosome complex; (3) Polyadenylation adds a poly-A tail at the 3' end by poly(A) polymerase, which protects the mRNA from degradation and aids in ribosome recruitment. These modifications ensure proper mRNA stability, nuclear export, and efficient protein synthesis in the cytoplasm.

Eukaryotic pre-mRNA undergoes three major processing steps: 5' capping, polyadenylation, and splicing. The 5' cap is added co-transcriptionally when the first 20-30 nucleotides have been synthesized. The cap is a modified guanine nucleotide (7-methylguanosine) that protects the RNA from degradation, facilitates nuclear export, and is recognized by ribosomes for translation initiation. Polyadenylation occurs at the polyadenylation signal (AAUAAA) in the 3' UTR. RNA polymerase II continues transcribing past this signal, and the pre-mRNA is cleaved at a site approximately 10-30 nucleotides downstream. A poly(A) tail (approximately 250 adenine residues) is then added by poly(A) polymerase. The poly(A) tail protects the mRNA from degradation, facilitates nuclear export, and enhances translation efficiency.