Long non-coding RNAs (lncRNAs) constitute a class of RNA molecules exceeding 200 nucleotides in length. Unlike coding RNAs, they do not serve as templates for protein synthesis. These lncRNAs are prevalent in both the animal and plant kingdoms. Initially regarded as mere byproducts of RNA polymerase II (Pol II) transcription—a genomic "noise" lacking biological relevance—lncRNAs have emerged from this shadow through extensive subsequent research.

Numerous investigations have unveiled the active roles of lncRNAs in diverse regulatory processes within organisms. This spans activities such as orchestrating chromatin remodeling, influencing RNA splicing, and modulating translation. Remarkably, lncRNAs can even serve as intricate scaffolds or reservoirs for microRNAs, contributing to intricate regulatory networks. However, the full extent of lncRNA functions remains largely uncharted.

An overarching assessment of lncRNAs and mRNAs on a global scale indicates that lncRNA genes tend to be less evolutionarily conserved. They typically exhibit fewer exons and lower expression levels. The diminished expression of lncRNAs might correlate with the presence of suppressive histone modifications at gene promoters. Furthermore, a considerable portion of identified lncRNAs exhibit conserved secondary structures, specific splicing patterns, and localized positions within cells.

LncRNA Classification

Tens of thousands of long non-coding RNA (lncRNA) motifs have been meticulously identified across a spectrum of organisms, encompassing unicellular eukaryotes to humans. These lncRNAs have been systematically classified into five distinct categories based on their precise location in relation to protein-coding genes:

lncRNA classification according to their orientation and position in the genome. (Latgé et al., 2018)

• Intergenic lncRNA (lincRNA): These transcripts originate from DNA sequences nestled between two protein-coding genes.
• Intronic lncRNA (intronic lncRNA): Emerging from within an intron of a protein-coding gene, this class of lncRNAs represents a noteworthy category.
• Sense lncRNA: These lncRNAs are transcribed in parallel with the direction of the protein-coding gene, forming an intriguing juxtaposition.
• Antisense lncRNA: In stark contrast, antisense lncRNAs are transcribed in the opposing direction to that of the protein-coding gene, creating a distinctive regulatory dynamic.
• Bidirectional lncRNA: A unique subset among lncRNAs, bidirectional lncRNAs are remarkable in that they share a common promoter with protein-coding genes but exhibit transcriptional activity in the opposite direction, emphasizing their regulatory complexity.

Mechanisms of LncRNA

Transcriptional Regulators

Long non-coding RNAs (LncRNAs) play a pivotal role in transcriptional regulation. They exhibit both cis-acting and trans-acting functions, not only influencing gene expression near their transcriptional origins (cis function) through diverse mechanisms but also orchestrating genome-wide gene expression and affecting gene localization by targeting distant transcriptional activators or repressors (trans function).

Chromatin Regulation

The intricate landscape of LncRNA-mediated chromatin regulation has been unveiled through chromatin conformation capture techniques coupled with RNA-chromatin association assays. LncRNAs, owing to their negatively charged nature, can counterbalance positively charged histone tails, resulting in chromatin de-condensation. Mechanistically, both cis- and trans-nuclear lncRNAs can engage with DNA to reshape the chromatin landscape. The specific roles encompass:

• Localization and Functional Impact of Protein-lncRNA Complexes on Chromatin.
• Direct Interactions between LncRNAs and DNA.

Nuclear Organization

LncRNAs assume a crucial role in nuclear organization by contributing to membrane-free RNA-protein phase separation within cells. They act as scaffolds or regulators, playing an indispensable role in the formation and functionality of diverse nuclear condensates.

Post-Transcriptional Regulation

LncRNAs exert post-transcriptional regulation through several modes:

• Direct Interaction with Proteins: LncRNAs form specific complexes with proteins, known as lncRNA protein complexes (lncRNPs), by binding to RNA sequence motifs or structures. These complexes subsequently modulate mRNA splicing, transcription, and signaling pathways in specific biological contexts. This mechanism may also involve post-translational modifications of splicing factors regulated by lncRNAs.
• Pairing with Other RNAs: Certain lncRNAs can directly base-pair with other RNA molecules, recruiting protein complexes involved in mRNA degradation. Notably, trans-lncRNAs have emerged as significant post-transcriptional regulators.
• MiRNA "Sponges": Some lncRNAs contain miRNA complementary sites and act as competing endogenous RNAs (ceRNAs). They regulate gene expression by sequestering miRNAs, reducing their targeting of mRNAs, and thus influencing subsequent target mRNA expression.

Organelle Regulatory

Remarkably, many lncRNAs exhibit specific subcellular localization within organelles, including exosomes and mitochondria. Exosome-localized lncRNAs can be secreted into recipient cells, contributing to epigenetic inheritance, cell-type reprogramming, and regulation of genomic stability. In contrast, mitochondria-localized lncRNAs, encoded by both nuclear and mitochondrial DNA, are associated with diverse functions, including mitochondrial metabolism, apoptosis, and interplay between mitochondrial and nuclear processes. Further research is needed to elucidate the precise functions and molecular mechanisms of organelle-specific lncRNAs.

Long Non-Coding RNA (lncRNA) Sequencing Technology

Rapid Amplification of cDNA Ends Sequencing (RACE-seq)

RACE-seq represents a groundbreaking technique in molecular biology, particularly for exploring the intricate world of long non-coding RNAs (lncRNAs). This method leverages reverse transcription and polymerase chain reaction (PCR) to elucidate the elusive portions of a gene transcript, ultimately yielding the complete mRNA sequence (cDNA). In essence, RACE-seq simplifies and accelerates the generation of full-length cDNAs, specifically targeting the 5' and 3' ends of low-abundance transcripts. This approach boasts remarkable speed, convenience, and efficiency, allowing for the simultaneous identification of multiple transcripts. Consequently, in recent years, RACE-seq has emerged as a superior alternative to traditional cDNA library screening methods, establishing itself as the preferred avenue for cloning full-length cDNA sequences.

• 3'RACE

In the 3' RACE (Rapid Amplification of cDNA Ends) technique, the poly(A) tail located at the 3' end of the mRNA serves as the starting point. An Oligo(dT)30MN molecule, attached to a universal primer designed for SMART oligonucleic acid sequences, functions as a 'locking primer' to initiate the synthesis of the first-strand cDNA through reverse transcription. Subsequently, a gene-specific primer, known as GSP1 (GSP), acts as the upstream primer, while a universal primer (UPM), containing a portion of the junction sequence, serves as the downstream primer. The first-strand cDNA, produced earlier, becomes the template for a series of PCR cycles, ultimately amplifying the DNA fragment located at the 3' end of the target gene.

• 5'RACE

The 5'RACE (5' Rapid Amplification of cDNA Ends) technique begins by using the poly(A) tail at the 3' end of the mRNA as a starting point. We employ Oligo(dT)30MN as a 'locking primer' to initiate the synthesis of the first-strand cDNA using reverse transcription, powered by the MMLV reverse transcriptase enzyme.

What sets MMLV apart is its terminal transferase activity, which adds 3-5 (dC) residues automatically when it reaches the 5' end of the first cDNA strand. These added (dC) residues then bind to a universal junction primer containing the SMART oligonucleotide sequence Oligo(dG), effectively converting it into a SMART sequence as part of the 5'RACE process. After this annealing step, further extension using the SMART sequence as a template transforms the (dC) residues into a universal junction.

Moving forward, we use a universal primer (UPM) that includes a part of the junction sequence as the upstream primer. For the downstream primer, we employ a gene-specific primer 2 (GSP2) tailored to the specific target gene. A PCR cycle is carried out, using the SMART first-strand cDNA as the template, to amplify the 5' end of the target gene, resulting in a cDNA fragment.

In the end, by analyzing and designing primers that match the overlapping sequences found in the 3' and 5' RACE products, we can ultimately obtain full-length cDNA. This innovative approach allows for a comprehensive understanding and characterization of gene sequences.

RIP Sequencing

The RNA Binding Protein Immunoprecipitation (RIP) technique is a valuable method employed to delve into the intricate interactions between RNA molecules and proteins within cells. This method utilizes specific antibodies that target the protein of interest, allowing for the precipitation of the associated RNA-protein complexes. Subsequently, the isolated and purified RNA bound to these complexes is subjected to analysis. In essence, RIP employs antibodies or epitope markers to selectively capture endogenous RNA-binding proteins residing in the cell's nucleus or cytoplasm, effectively preventing nonspecific RNA binding.

To ensure the accuracy of the results, it is imperative that the reagents and antibodies employed in the RIP reaction system are devoid of RNA enzymes. Additionally, the antibodies used must undergo validation through RIP experiments. The subsequent steps involve the separation of the RNA-binding protein from its bound RNA through the process of immunoprecipitation. The sequences of the bound RNA are then identified through techniques such as microarray analysis (RIP-Chip), quantitative RT-PCR, or high-throughput sequencing (RIP-Seq).

RIP serves as a robust and indispensable tool for unraveling the dynamic processes inherent to post-transcriptional regulatory networks. It plays a pivotal role in uncovering the regulatory targets of miRNAs, offering invaluable insights into the intricacies of gene expression control.

Reference:

1. Latgé, Guillaume, et al. "Natural antisense transcripts: molecular mechanisms and implications in breast cancers." International journal of molecular sciences 19.1 (2018): 123.