Introduction
Small RNAs play regulatory roles of downstream genes and modulate chromatin structure of the genome. There are many families of small RNAs being the most widely known microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs play a role in a broad rage of biological process including development, cell differentiation and has been associated with human diseases such as cancer. MicroRNAs can reduce the activity (expression level) of protein coding genes through inhibition of protein translations and destabilisation of the mRNAs via removal of the poly A tail.
What is small RNAseq?
Small noncoding RNAs act in gene silencing and post-transcriptional regulation of gene expression. Small RNA sequencing (RNA-Seq) is a technique to isolate and sequence small RNA species, such as microRNAs (miRNAs). Small RNA-Seq can query thousands of small RNA and miRNA sequences with unprecedented sensitivity and dynamic range (Source: Illumina).
With small RNA-Seq you can discover novel miRNAs and other small noncoding RNAs, and examine the differential expression of all small RNAs in any sample. You can characterize variations such as isomiRs with single-base resolution, as well as analyze any small RNA or miRNA without prior sequence or secondary structure information (Source: Illumina).
Key Classes of Small RNAs:
MicroRNAs (miRNAs): miRNAs are approximately 21-24 nucleotides in length and are processed from longer precursor transcripts. They regulate gene expression by binding to complementary sequences in target mRNAs, usually resulting in mRNA degradation or translational repression. MiRNAs are involved in numerous biological processes, including development, cell differentiation, stress responses, and disease pathways like cancer.
Small Interfering RNAs (siRNAs): siRNAs are similar in size to miRNAs but are often derived from double-stranded RNA (dsRNA) precursors. They play key roles in the RNA interference (RNAi) pathway, where they guide the RISC to cleave specific target mRNAs, resulting in their degradation. SiRNAs can be endogenous or exogenously introduced and are often involved in antiviral responses and gene knockdown experiments.
Piwi-Interacting RNAs (piRNAs): PiRNAs are slightly longer, ranging from 24-30 nucleotides, and interact with the PIWI subfamily of Argonaute proteins. They are primarily expressed in the germline and play essential roles in maintaining genomic integrity by silencing transposable elements.
Animal and plant microRNAs - similarities and differences
Similarities:
Biogenesis and Processing:
Both animal and plant miRNAs are derived from longer precursor transcripts called primary miRNAs (pri-miRNAs). These pri-miRNAs are processed into precursor miRNAs (pre-miRNAs) and finally into mature miRNAs.
In both cases, the mature miRNAs are incorporated into an RNA-induced silencing complex (RISC) to carry out gene silencing functions.
Role in Gene Regulation:
In both animals and plants, miRNAs regulate gene expression primarily at the post-transcriptional level. They bind to complementary sequences in their target mRNAs to either induce degradation or inhibit translation. • They play crucial roles in controlling a wide range of biological processes such as development, stress responses, and cell differentiation.
Conservation in Function:
Some miRNAs, such as those involved in essential cellular processes like growth and differentiation, are conserved across both plants and animals. For instance, the miR-156/157 family in plants and the let-7 family in animals are key regulators of developmental timing.
Differences:
| Animals | Plants |
|---|---|---|
Target recognition and binding mechanism | miRNAs often recognize target mRNAs through imperfect base pairing, particularly in the 3’ untranslated region (3’ UTR) of the mRNA. The “seed region” (nucleotides 2-8) at the 5’ end of the miRNA plays a crucial role in target recognition. Due to the imperfect base pairing, a single miRNA can regulate many target genes. | miRNAs generally have near-perfect or perfect complementarity to their target mRNAs, usually within the coding region. This leads to direct cleavage and degradation of the target mRNA. Plant miRNAs often have a one-to-one relationship with their targets. |
Biogenesis and processing pathways | The primary miRNA (pri-miRNA) is processed in the nucleus by the Drosha-DGCR8 complex into a pre-miRNA. The pre-miRNA is then exported to the cytoplasm, where it is further processed by the enzyme Dicer to produce the mature miRNA. | Both the processing of pri-miRNAs into mature miRNAs and the loading into RISC take place mainly in the nucleus. The enzyme Dicer-like 1 (DCL1) is responsible for cleaving the pri-miRNA and pre-miRNA in a single-step process. |
RISC Composition and Argonaute Proteins | The RISC is typically composed of an Argonaute protein (AGO1 being the most common) and the miRNA. There are multiple AGO proteins with diverse functions. | While AGO1 is the predominant Argonaute protein in plant miRNAs, some plants have additional AGO proteins with specialized roles, such as AGO2 and AGO7. |
Function and Location of Target Sites | miRNAs usually target the 3’ UTR of mRNAs, leading to translational repression or, less commonly, mRNA degradation. Rarely, they can target the 5’ UTR or coding regions. | miRNAs predominantly target the coding regions or the 5’ UTRs of mRNAs, which often results in mRNA cleavage. |
Length and Structure of Precursor miRNAs | Precursor miRNAs are typically 70-100 nucleotides long and have a characteristic hairpin structure. | Precursor miRNAs tend to be longer, ranging from 70 to 200 nucleotides, and the hairpin structures are often more variable. |
Evolutionary Conservation | miRNAs are highly conserved across species, reflecting their critical roles in regulating fundamental biological processes. | While many miRNAs are conserved among closely related plant species, there is generally less conservation at broader taxonomic levels. |
Overall characteristics
In Mammals: AGO2 is the key catalytic AGO protein with slicing activity, while AGO3 and AGO4 are primarily involved in translational repression and gene regulation.
In Plants: AGO2 functions in antiviral defense, AGO4 and AGO6 are central to RNA-directed DNA methylation, AGO5 regulates reproductive tissues, and AGO7 plays an essential role in the ta-siRNA pathway and leaf development.
The diversity in AGO proteins reflects the specialized functions that have evolved in both animals and plants to fine-tune gene expression through small RNA-mediated pathways.
Advantages of Small RNA Sequencing
Generate miRNA sequencing libraries directly from total RNA to understand the role of noncoding RNA.
Understand how post-transcriptional regulation contributes to phenotype
Identify novel biomarkers
Capture the complete range of small RNA and miRNA species
Global community for nextflow Bioinformatics pipelines
nf-core is a community-led project to develop a set of best-practice pipelines built using Nextflow. Pipelines are governed by a set of guidelines, enforced by community code reviews and automatic linting (code testing). A suite of helper tools aim to help people run and develop pipelines.
nf-core small RNAseq pipeline https://nf-co.re/smrnaseq/2.2.3
Let’s now go to hands on excercises: