8.2: Nucleic Acids - RNA Structure and Function

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Learning Goals (ChatGPT o1, 1/30/25)

1. Differentiate RNA from DNA:

   • Describe the key chemical differences (e.g., ribose vs. deoxyribose; uracil vs. thymine) and explain how these differences influence the biological functions of RNA.

2. Analyze RNA Structural Complexity:

   • Explain how RNA can form secondary (e.g., stem-loop, hairpin) and tertiary structures, and evaluate the role of noncanonical base pairing and chemical modifications in these processes.

3. Understand RNA Transcription and Processing:

   • Illustrate the process of transcription from DNA to RNA, including the formation of heteronuclear RNA and subsequent splicing events that yield mature mRNA.

4. Classify RNA Types Based on Function and Size:

   • Distinguish between coding RNAs (e.g., mRNA) and noncoding RNAs, and further classify noncoding RNAs into short (<200 nt) and long (>200 nt) categories with examples such as tRNA, rRNA, miRNA, snoRNA, lncRNA, and circRNA.

5. Evaluate the Role of RNA in Protein Synthesis:

   • Describe the functions of messenger RNA in coding for proteins and the roles of transfer RNA and ribosomal RNA in translation, including the molecular interactions that facilitate these processes.

6. Investigate RNA–Protein Interactions:

   • Analyze specific examples of RNA–protein complexes (e.g., ribosome components, spliceosomal snRNPs, toxin-antitoxin systems) to understand how RNA structure and binding partners influence cellular functions.

7. Explore RNA-Mediated Gene Regulation:

   • Explain how noncoding RNAs such as microRNAs and siRNAs regulate gene expression through mechanisms like mRNA degradation, translational inhibition, or transcriptional modulation.

8. Apply Thermodynamic Concepts to RNA Folding:

   • Interpret the concept of RNA’s thermodynamic folding landscape and compare it to protein folding, discussing how environmental factors and protein binding can lead to dynamic structural rearrangements.

9. Examine RNA’s Role in Cellular and Disease Contexts:

   • Discuss the significance of RNA molecules in cellular processes (e.g., splicing, ribosome function, synaptic regulation) and their emerging roles in diseases such as cancer, cardiovascular disorders, and neurological conditions.

10. Integrate Bioinformatics and Structural Tools:

    • Utilize available computational programs and databases (e.g., lncipedia, miRBase, CircAtlas) to predict RNA secondary structures and explore experimentally determined RNA–protein complexes via interactive 3D models.

Each goal is designed to promote critical thinking, link structure to function, and encourage practical engagement with current research tools in RNA biology.

RNA: Structure and Function

Ribonucleic acids are very similar in chemical structure to DNA, except they contain ribose instead of deoxyribose. They also have the pyrimidine base uracil instead of thymine, as shown in Figures 1 and 2 above. These two small changes (but mostly the first) confer on it a very different set of biological functions than DNA. This should not surprise us, and the basis of all chemistry and biochemistry is that chemical structure determines chemical and biochemical functions and activities. In the previous section, we discussed how RNA can adopt complex tertiary structures, which requires the presence of more noncanonical base pairs and chemical modification of bases. In this section will explore the plethora of different types of RNA structures and their functions.

The sequence of RNA is made from DNA through a process called transcription (converting the information of DNA, a nucleic acid, into RNA, another nucleic acid). RNA can form double-stranded helices, but typically, these are viral in origin. DsRNA is a pathogen-associated molecular pattern (PAMP) that binds Toll-like receptor 3 (TLR3), as seen in Chapter 5.5. If both strands of DNA are transcribed, the resulting strands can anneal form dsRNA. In addition, a single strand of RNA can fold on itself if the 5' and 3' ends are complementary to form a stem-hairpin loop. Figure \(\PageIndex{1}\) shows a stem-loop from a messenger RNA (4QOZ) when it is bound to a specific RNA binding protein (not shown).

Figure \(\PageIndex{1}\) A stem-loop from a messenger RNA (4QOZ) when it is bound to a specific RNA binding protein (not shown).

Larger ss-RNA can form tertiary structures with many regions of intrastrand hydrogen bonds forming secondary structures, as shown in Figure \(\PageIndex{2}\) for one type of RNA called a transfer RNA

Figure \(\PageIndex{2}\): Secondary and tertiary structure of tRNA showing unpaired (gray) and hydrogen-bonded secondary structure regions (color). Kyle Schneider https://commons.wikimedia.org/w/inde...curid=12309266

Figure \(\PageIndex{3}\) a computed model for secondary structure within a much larger single RNA molecule S11 that is part of the ribosome. The figure shows color-coded differences in accessibility when the S11 RNA is free (blue) and bound to the protein NSP2 (red), which induces structural rearrangements.

Figure \(\PageIndex{3}\): Model showing secondary structure on ribosomal S11 RNA and accessibility changes on binding the protein NSP2.

You can imagine that a different set of intrachain H-bonded double-stranded regions could easily form, with the most likely determined by sequence, local environment, and protein binding partners. Each RNA molecule would have a thermodynamic folding landscape similar to that of protein. Programs are available to determine secondary structures from RNA sequences. Each RNA molecule would have a thermodynamic folding landscape similar to that of protein. Also, the structures are dynamic, as we can see with proteins.

Another feature that complicates RNA is that many different types of RNA are made from DNA using RNA polymerases. They are loosely divided into two types of RNA. One is coding RNA, which contains the sequence information that will be translated into a protein sequence. The other type is called noncoding RNA. These RNAs regulate many cellular processes, including transcription which produces coding RNA.

Coding RNA

The DNA template from which the coding sequence of a translatable RNA is produced is called a gene. The coding RNA, which has the exact sequence that is translated into protein, is called messenger RNA (mRNA). The exact sequence of RNA in mRNA that encodes a protein is derived from a longer contiguous DNA sequence in the nucleus from which sections called intervening sequences or introns have been removed. The coding sequences of DNA, which are separated by introns, are called exons. When coding DNA is transcribed, a long contiguous sequence containing both exons and introns is transcribed into one long primary transcript called heteronuclear RNA. The introns in the heteronuclear RNA are removed in a splicing reaction catalyzed by a large complex called the spliceosome to form mRNA. The process is illustrated in Figure \(\PageIndex{4}\). The first RNA sequence made is the heteronuclear RNA.

Figure \(\PageIndex{4}\): https://commons.wikimedia.org/wiki/F...ns_introns.gif. This work is in the public domain in the United States

The long single-stranded mRNA molecule binds to ribosomes and nanomachines, which orchestrate the translation of the mRNA sequence into a protein sequence. Around 20,000 human genes produce an even larger number of mRNA that arise from differential splicing of the primary transcript.

Noncoding RNA (ncRNA)

Not long ago, few thought about possible RNA transcripts from non protein-coding regions of the genome, except for two types of RNA required to translate mRNA. These two are ribosomal RNAs (rRNA) found in ribosomes and transfer RNAs, to which amino acids are esterified and transferred to a growing protein chain of the ribosome. Many more classes have been discovered and given names that confuse someone more used to protein structures. One way to classify noncoding RNAs (ncRNAs) is based on size.

• short noncoding RNAs (sncRNAs) are <200 nucleotides
• long noncoding RNAs (lncRNAs)are >200 nucleotides

These function to regulate gene expression at both the transcription and post-transcriptional levels. Some have catalytic functions. Some affect chromosome structure and chemical modification.

Long Noncoding RNAs (lncRNAs)

There may be between 16,000 to over 100,000 human lncRNAs encoded into the genome, which adds much complexity to our understanding of the function of RNA transcripts. An online lncipedia is a database of searchable lncRNA sequences. There are many types of lncRNAs. The first we will consider is ribosomal RNA.

a. Ribosomal RNA (rRNA):

These RNAs fit the simple definition of lnRNAs (>200 nucleotides and are not protein- ing), but most would not think of them as lncRNAs since they have always been in their own category of a nonprotein-coding gene. rRNAs vary in length from between 1500 and 3000 nucleotides long in bacteria and about 1800 and 5000 nucleotides long in humans and are the core structure of ribosomes, the nanomachines that translate bound mRNA into a protein sequence.

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the structure of 23S rRNA of the large ribosomal subunit from Deinococcus radiodurans (2O44) (long load time).

Figure \(\PageIndex{5}\): Structure of 23S rRNA of the large ribosomal subunit from Deinococcus radiodurans (2O44). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?Tb2BVwSTe6LviPou5

The red (highlighted yellow) spacefill is the 5' start of the rRNA. The chain has a complex tertiary structure, like a protein sequence, and ends at the cyan spacefilling 3' end. It has 2880 nucleotides.

b.  Other types

Let's focus on more classic examples of long noncoding RNAs (i.e., not rRNA). One way to categorize them is based on the position in the genome that encodes them. The different types include long intergenic noncoding RNAs (lincRNA), intronic lncRNAs, antisense RNAs (as lncRNAs), and other variants. These are illustrated in Figure \(\PageIndex{6}\), where the lncRNA is pink.

Figure \(\PageIndex{6\): https://en.Wikipedia.org/wiki/Long_non-coding_RNA. Creative Commons Attribution-Share Alike 3.0 Unported

Another variant is exon and intron-containing circRNAs (EIciRNAs), as illustrated in panel B in Figure \(\PageIndex{13}\). These are presumably produced from pre-mRNA for a given mRNA and appear to regulate gene expression through RNA-RNA interactions with U1 snRNA, which starts the assembly of the spliceosome on pre-mRNA when it binds to the 5′ pre-mRNA splice site.

circRNA are found throughout the biological world and have been associated with diseases such as cancer, cardiovascular disease, and brain disorders like Alzheimer's.  Their possible functions include binding to miRNAs and RNA binding proteins, altering their activities, and DNA, regulating its transcription.  CircAtlas is a database of circRNAs that have been experimentally validated by two techniques

Now let's consider specific examples of long non-coding RNAs (lncRNAs), which are often bound to target proteins.

• mamRNA (a lnc RNA)

The lncRNA named mamRNA (Mmi1 and Mei2-associated RNA) binds two proteins, Mmi1 and Mei2 in Schizosaccharomyces pombe that control the balane between meisois and mitosis in yeast. (Schizosaccharomyces pombe is a "fission" yeast that divides by fission, not budding.) The MamRNA has two variants of length 550 and 700 nucleotides. The binding of mamRNA leads to the ubiquitinylation of the Mei2 in the complex. Mmi1 R is an RNA-binding protein that binds to a modified version of adenosine methylated at N6 and is found internally in mRNA. Mei2 (meiosis protein 2) is necessary of meiosis. The binding of mamRNA leads to the ubiquitinylation of the Mei2 in the complex, targeting it for proteolysis. Mei2 concentrations relatively increase, shifting yeast from mitosis to meiosis. Figure \(\PageIndex{7}\) shows a cartoon depicting these interactions.

Figure \(\PageIndex{7}\): Binding of the lncRNA mam2 to yeast proteins Mei2 and Mmi1. Andric, V. et al. Yeast. Non-coding RNA 2021, 7, 34. https://doi.org/10.3390/ncrna7020034. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of the S. pombe Mei2 RRM3 protein domain bound to the Mei2 binding "domain" of mamRNA (6YYM), which in this structure is only eight nucleotides long (not the full length this lncRNA which is 550 and 700 nucleotides long).

Figure \(\PageIndex{8}\): S. pombe Mei2 RRM3 protein domain bound to an eight nucleotide section of the yeast lncRNA mam (6YYM). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...5xmQjwAmQL9on7

• ToxI - a lnRNA inhibitor of the endonuclease ToxN

Those with a more chemistry-centric background might be surprised that viruses also "infect" bacteria. These viruses are called bacteriophages. It is estimated that there are over 10³⁰ in nature. Some covalently incorporate into genomes where they reside and are incorporated permanently into the genome. They are a main driver of bacterial genome evolution as they shape the bacteria's immune response and adaptation.

One very interesting example is the type III toxin-antitoxin (TA) system in E. Coli. It consists of a toxin, ToxN, which is a nuclease that cleaves internally after the second A in a AAA sequence. It acts on mRNA, but especially pre-mRNA sequences. It is inhibited by the binding of a lncRNA called ToxI (toxin inhibitor). The RNA sequence of the ToxI inhibitor has 36 "domain" repeats of a pseudoknot, one of which is sufficient to inhibit the ToxN. The ToxN endonuclease cleaves the ToxI lncRNA as it assembles the complex. It also cleaves its mRNA. Figure \(\PageIndex{9}\) shows an interactive iCn3D model of the protein toxin (ToxN):lncRNA (ToxI), which is a shortened version of 29 nucleotide section from Pectobacterium atrosepticum (2xdb).

Figure \(\PageIndex{9}\): Protein toxin (ToxN) - 29 NT fragment of lncRNA antitoxin ToxI complex (2xdb). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...KjNPGghiUyAXr9

As we discussed in Chapter 3.2:  The Structure of Proteins- An Overview, some long noncoding RNAs have noncanonical open reading frames (ncORFs) that are translated into protein, adding an element to the complexity of RNA.

Short Noncoding RNA

Short noncoding RNAs (sncRNAs) are less than 200 nucleotides in length. By definition, this would include transfer RNAs (tRNAs), which bring to the ribosome amino acids covalently attached to their 3' end of the tRNA for incorporation into a growing protein chain during the translation of mRNA. As with rRNA for lncRNAs, these are really in a class of their own. Others include small nuclear RNAs (snRNAs) involved in splicing, small nucleolar RNAs (snoRNAs) involved in the modification of rRNAs, and microRNAs (miRNAs), involved in the inhibition of translation and transcription, PIWI-interacting RNAs (piRNAs), and endogenous small interfering RNAs (siRNAs). It is difficult to remember the subtle differences among these, making them difficult to understand. We will tell their stories with a few targeted examples.

a. Transfer RNA:

Transfer RNAs act as adapter molecules between transcription and translation. They are between 76 and 90 nucleotides long and have a cloverleaf shape. An enzyme, aminoacyl-tRNA synthase, covalently attaches a select amino acid at its 3' end. Another end of the tRNA hydrogen bonds through 3 nucleotides (the anticodon) to a triplet nucleotide (the codon) on the mRNA that encodes a specific amino acid at that triplet position. Figure \(\PageIndex{10}\) shows an interactive iCn3D model of the structure of yeast phenylalanine tRNA (1EHZ)

Figure \(\PageIndex{10}\): Yeast phenylalanine tRNA (1EHZ) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?iAWpKknDWbQm5hHL6

b. Small nuclear RNA (snRNA):

The spliceosome is a nanoparticle that catalyzes the removal of introns from pre-mRNA in eukaryotes (prokaryotes appear devoid of introns). The yeast spliceosome has a molecular weight of 1.3 million and contains five small ribonucleoproteins (RNPs) with many other associated proteins. Each of the 5 RNPs has a small nuclear RNA (U1, U2, U4, U5, and U,6) enriched in uracils. U6 is highly conserved and is directly involved in catalysis. Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the core structure of the U6 small nuclear ribonucleoprotein complex with most of the U6 RNA bound.

Figure \(\PageIndex{11}\): Core structure of the U6 small nuclear RNA - protein (ribonucleoprotein) complex (4N0T). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...P3SLVRAKhP7W57

c. MicroRNAs (miRNAs) and small inhibitory RNAs (siRNAs)

MicroRNAs (miRNAs) control the expression of thousands of genes in plants and animals. They are single-stranded but fold on themselves to form a stem-hairpin. The miRBase is a microRNA database containing almost 40,000 miRNA sequences. miRNAs are highly conserved and are found in animals, plants, and some unicellular eukaryotes. They interact with the 3′ untranslated regions of mRNAs and inhibit or prevent their translation. Several key proteins, RNA polymerase II, Drosha and Dicer are involved in the canonical pathway while the others appear to be independent of Drosha, which is a ribonuclease III double-stranded (ds) RNA endoribonuclease.

Dicer is a dsRNA) endoribonuclease, which cleaves long dsRNAs and short hairpin pre-microRNAs (miRNA) into fragments of either 21-23 nucleotides (short interfering RNA) or 19-25 nucleotides (microRNAs). Each has two nucleotides that are unpaired at the 3' end. These bind to the enzyme complex RISC ( RNA-induced silencing complex), which then targets them to mRNA complementary to the siRNA/miRNA (RISC), causing cleavage of the mRNA and hence inhibiting translation.

Small inhibitory RNAs (siRNAs) are very similar to miRNA (to the point that differentiating between them is somewhat arbitrary). They both engage in RNA interference (RNAi) of mRNA translation. Here are some reported differences:

• The substrate for dicer cleavage is dsRNA (that could be added exogenously) of length 30-100+ for siRNA, but the actual pre-miRNA of length 7-100 nucleotides that may contain hairpins with some mismatches (i.e., not a perfect stem and hairpin)
• The final RNA after dicer processing is double-stranded for both and 21-23 nucleotides long for siRNA and 19-25 for miRNA
• siRNAs are perfectly complementary to the target mRNA,s while miRNAs, which are not necessarily perfectly complementary, bind typically to the 3' untranslated end of the mRNA
• Because of the perfect complementarity to target mRNA, siRNA interact with only one mRNA while miRNAs, given that they are not perfectly complementary to their target sequences, can bind different mRNAs
• Given their higher affinity binding, the siRNA leads to dicer endonuclease cleavage of the target mRNA. In contrast, inhibition of mRNA translation by miRNAs arises from binding of the miRNA to the mRNA or, if the match between the miRNA and mRNA is high enough, endonuclease cleavage of the mRNA.

Figure \(\PageIndex{12}\) shows canonical and several alternative pathways for their transcription and processing from the noncoding miRNA genes.

Figure \(\PageIndex{12}\):

Figure 1. Canonical and non-canonical pathways of microRNA biogenesis. (A) Canonical pathway—microRNA gene is transcribed by RNA polymerase II into primary microRNA (pri-miRNA), cleaved by microprocessor complex Drosha/DGCR8, and precursor microRNA (pre-miRNA) is exported from the nucleus to the cytoplasm by Exportin 5 (XPO5) and further processed by Dicer and its partners into 18–25 nucleotide long microRNA duplex with 2-nucleotide 30 overhangs. The guide strand is subsequently bound by the Argonaute proteins 1-4 (AGO1-4) and retained in the microRNA-induced silencing complex to target mRNAs for post-transcriptional silencing. (B) Mirtrons—generated through mRNA splicing independently of the Drosha-mediated processing step. (C) Small nucleolar RNA-derived microRNAs—Drosha-independent pathway. (D) Exportin 5-independent transport of pre-miRNAs from the nucleus to the cytoplasm has been described in the case of miR-320 family. (E) Dicer-independent processing of miR-451—pre-miR-451 is directly loaded into AGO2, cleaved, and trimmed by poly(A)-specific ribonuclease PARN to produce mature miR-451. Gregorova et al. Cancers 2021, 13, 1333. https://doi.org/10.3390/cancers13061333. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

siRNA that are perfect matches to specific mRNA can be easily designed and purchased for translation inhibition and through gene silencing studies. Both miRNAs and siRNA are potentially therapeutic as it is much simpler to design a drug that targets a mRNA sequence (a 1D sequence target) than a protein active site (a 3D target). Additionally, they can be used to inhibit protein synthesis of target proteins that don't have a "druggable" active site.

The protein Argonaute is involved in miRNA and siRNA silencing of genes through their mRNAs in the RISC ( RNA-induced silencing complex). RISC contains the protein argonaute 2 (AGO2) bound to a "guide" RNA, which is either microRNA (miRNA) or short interfering RNA (siRNA). The miRNA or siRNA directly interacts with the "target" - the mRNA.

• Example miRNA:

Figure \(\PageIndex{13}\) shows an interactive iCn3D model of human Argonaute2 Bound to a Guide (miRNA) and Target RNA (4W5O). The two RNA sequences are 5' UUCACAUUGCCCAAGUCUUU 3' and 5' CAAUGUGAAA 3'.

Figure \(\PageIndex{13}\): human Argonaute2 Bound to a Guide and Target RNA (miRNA) (4W5O). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?9fvyQmPkk4Kctei59

Recent Updates:  October 9, 2024

2024 Nobel Prize in Physiology and Medicine for discovery of miRNAs

Victor Ambros and Gary Ruvkun won the Nobel Prize (October 7, 2024) for discovering miRNA and its function in the roundworm C. elegans, which has fewer than 1000 cells.  Their basic research on miRNA's role in protein synthesis regulation was extended to other organisms.  Humans express over 1000 miRNAs that regulate translation.  

Ruvkun found that two small RNAs, lin-4 (22 nucleotides) and let-7 (21 nucleotides),, are required to progress from larval to adult stages in the worm.   They were nonhomologous but complementary to parts of the 3'-untranslated region (UTR) of mRNAs for proteins that are downregulated in development.  Let-4 is found in many animal species, including humans, and binds to the 3'-UTR of the mRNA for the protein lin-41. Ambrose discovered that a small RNA lin-4 from a non-protein encoding gene was complementary to regions in the 3'-UTR of the mRNA for a protein lin-14.

The seed sequences of miRNAs are a conserved 7-mer at positions 2-7 from the miRNA 5'-end. This part of the miRNA must be exactly complementary to the 3'-UTR of the target mRNA, but the other bases don't have to match exactly.

The sequence of let-7 miRNA from C. elegans is 5'-UGAGGUAGUAGGUUGUAUAGU-3'.  Figure \(\PageIndex{i}\) below shows the aligned sequences of a let-7 miRNA and lin-41 mRNA (A) and analogs made for NMR structure determination (B,C).  

Figure \(\PageIndex{i}\): Sequences of a let-7 miRNA and lin-41 mRNA (A) and analogs.

( A ) Schematic representation of the complex of let-7 miRNA with 3′-UTR of the lin-41 mRNA (LCS 2). The residues in green and blue match the sequences used in the monomolecular and dimeric constructs. ( B ) The 33-nt monomolecular RNA construct mimicking the complex. ( C ) The dimeric RNA construct. The numbering used in (B) has been preserved to facilitate comparing the two constructs. The residues originating from the lin-41 mRNA sequence are labeled by asterisk.  Cevec, M, Thibaudeau, C and Plavec, J.  Nucleic Acids Research, Volume 36, Issue 7, 1 April 2008, Pages 2330–2337, https://doi.org/10.1093/nar/gkn088.  Creative Commons CC-BY-NC license. 

Two analog structures preserved most of the matching sequences from the miRNA and mRNA (green for the let-7 miRNA and blue for the lin-41 mRNA.  The seed sequence of the miRNA was completely preserved. One was a single strand that formed a stem-loop structure. Figure \(\PageIndex{j}\) shows an interactive iCn3D model of the solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans (2JXV) using the single-stranded analog.

Figure \(\PageIndex{j}\): solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans (2JXV). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...xJh4paMJRiTQ47

G1 (red spacefill) is the 5'-end and C33 (yellow) is the 3'-end of the single-stranded analog. The green bases match those from the figure above. For clarity, the matching base partners of lin-41 mRNA are shown in cyan instead of blue.

The model shows two stems with an asymmetric loop containing 3 Us and 2 A.  A GU wobble base is seen in the first stem between U6 and G28.

 

• Example: siRNA (Small interfering RNA)

Virus genomes are ultimately decoded into new viruses by the host replication, transcription, and translation machinery. Host cells have evolved ways to silence mRNAs from viruses. Unfortunately, viruses, in response, evolve ways to suppress host RNA silencing. Many viral proteins are used by the host to suppress silencing. The viral p19 protein preferentially binds to host short interfering RNAs (siRNAs) than to microRNAs (miRNAs). A single mutation in the viral p19 proteins changes selectivity, allowing it to bind to a specific human miRNA called miR-122. This shows the subtle complexities of protein:RNA interactions. Figure \(\PageIndex{14}\) shows an interactive iCn3D model of the viral suppressor of RNA silencing protein and a 21 residue small interfering RNAs (6BJV)

Figure \(\PageIndex{14}\): Viral suppressor of RNA silencing protein p19 mutants and a double-stranded 21 nucleotide small interfering RNAs (6BJV). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VXzSgezTEPcPt9

• Example: piRNA (a specific miRNA)

Piwi proteins are RNA-binding proteins in plants and animals and are similar in structure to argonaute. They bind a guide RNA called piwi-interacting RNAs (piRNAs) and lead to the silencing of sequences called transposable elements that can move around the genome. piWi has endonuclease activity and can cleave mRNA. The piRNAs are just one type of miRNA. Figure \(\PageIndex{15}\) shows an interactive iCn3D model of Ephydatia fluviatilis (a sponge) PiwiA with a guide (piRNA) and-target RNA(7KX9)

Figure \(\PageIndex{15}\): Ephydatia fluviatilis PiwiA-piRNA-target complex (7KX9). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?j9ikApUgsVvqsiM5A

lncRNAs and miRNAs in the brain

After the decoding of the human genome, many have been struggling to understand how the complexity of the human brain (large size, greater connectivity among neurons) arises, given that we appear to have only around 20,000 protein genes encoded by the genome (not counting small proteins of less than 100 amino acids). Long noncoding RNAs (lncRNAs) and miRNAs appear significant pieces of this puzzle. Their ability to regulate transcription during development may hold the key. Additional roles on these RNAs outside of transcriptional regulation are being discovered. Some are transported away from the nucleus to serve other functions in axons, dendrites, etc. For example, the lncRNA Gm38257 binds to proteins that structure the synapse (components of the spectrin/ankyrin complex) instead of simply regulating gene expression.

The repertoire of miRNA appears to be significantly increased in "intelligent" organisms such as humans and octopi. For example, a large increase (179) in miRNAs occurs in proceeding in the evolutionary scale from mice (which have about 24,000 protein-encoding genes) to humans (around 20,000). miRNAs and lncRNAs may be involved (causative or correlative?) with brain disease. An example is the miR-124 is significantly elevated (3.5X) times higher in hippocampal cells from mouse models of Alzheimer's compared to normal mice. Altered expression of the lncRNA named Gomafu, RNCR2 or MIAT) appears to affect certain psychiatric diseases.

c. small nucleolar RNA

The nucleolus is a small nuclear structure that helps assemble the ribosomal RNAs synthesized in the nucleus. They then are transported through the nuclear membrane into the cytoplasm, where they combine with proteins translated from mRNA in the cytoplasm to form complete ribosomes. As described below, rRNA is chemically modified by enzymes (much like the post-translational modification of proteins). One such modification is 2'-O-methylation in archaea and eukaryotes. A class of small nucleolar RNAs (snoRNAs) that vary from 10-21 base pairs are called C/D RNAs, and they "guide" the modification. Hence, they are also called "guide" RNAs. These snoRNAs bind to 3-4 proteins into ribonucleoproteins. Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the box C/D ribonucleoprotein 40 nt snoRNA "guide" and a 10 nucleotide RNA target substrates. It appears that the maximal duplex RNA formed (from the guide and target) is 10 base pairs long.

Figure \(\PageIndex{16}\): Box C/D ribonucleoprotein with a small nucleolar RNA (snoRNA) 12 nucleotide "guide" and 13 nucleotide target RNA for 2'-O-methylation (5GIO). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?q45tA8LEko87mYTw6

Summary

This chapter provides an in-depth exploration of RNA’s unique chemical and biological roles, emphasizing how small differences in structure lead to diverse functions that are essential for cellular life.

Chemical Basis and Structural Diversity:
RNA differs from DNA primarily in that it contains ribose instead of deoxyribose and uracil in place of thymine. These seemingly minor differences not only alter its chemical properties but also empower RNA to adopt a wide range of structures—from simple stem-loop hairpins to complex tertiary conformations. Such structural variability is driven by noncanonical base pairing and chemical modifications, much like the dynamic folding observed in proteins.

Transcription and RNA Processing:
The chapter outlines the transcription process where RNA polymerases synthesize RNA from a DNA template. For coding RNAs, the initial heteronuclear transcript undergoes splicing to remove introns, resulting in mature messenger RNA (mRNA) that serves as the blueprint for protein synthesis. The intricacies of RNA processing are critical for ensuring that the correct genetic information is conveyed and translated.

Classification of RNA Types:
RNA molecules are broadly categorized into coding and noncoding RNAs. While mRNA carries the code for protein synthesis, noncoding RNAs (ncRNAs) serve regulatory and structural functions. The ncRNAs are further divided by size:

• Short noncoding RNAs (<200 nucleotides): Include tRNAs (adapters in translation), small nuclear RNAs (snRNAs) that are essential for splicing, microRNAs (miRNAs) and small interfering RNAs (siRNAs) involved in gene regulation, and other specialized molecules like piRNAs and snoRNAs.
• Long noncoding RNAs (>200 nucleotides): These include the well-known ribosomal RNAs (rRNAs) that form the core of the ribosome, as well as a diverse array of lncRNAs involved in transcriptional regulation, chromatin organization, and even direct protein interactions.

RNA–Protein Interactions and Functional Implications:
The chapter highlights numerous examples of RNA interacting with proteins to regulate cellular processes. From the assembly of the ribosome to RNA-based toxin-antitoxin systems in bacteria, these complexes underscore the importance of RNA’s structure in mediating its interactions. Detailed structural models illustrate how RNA–protein binding can affect processes like mRNA splicing, translation, and gene silencing.

RNA in Gene Regulation and Disease:
A significant portion of the discussion is dedicated to the roles of miRNAs and lncRNAs in regulating gene expression. These molecules control the translation and stability of mRNAs and are implicated in various diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. Notably, recent advances—highlighted by the awarding of the 2024 Nobel Prize in Physiology and Medicine—underscore the importance of miRNAs in developmental processes and disease pathogenesis.

Integration of Computational and Experimental Tools:
The chapter also emphasizes modern techniques used to predict RNA secondary structures and analyze RNA–protein complexes. Databases such as miRBase and lncipedia, along with interactive 3D structural models, allow biochemists to visualize the dynamic nature of RNA and its myriad interactions within the cell.

In summary, this chapter equips students with a comprehensive understanding of RNA's multifaceted roles—from its chemical foundation and diverse structural forms to its critical involvement in gene regulation and disease. Through the integration of molecular details and modern analytical tools, junior and senior biochemistry majors gain valuable insights into how RNA not only translates genetic information but also orchestrates a complex network of regulatory processes within the cell.

References

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