Primary microRNA, or pri-miRNA, is the initial transcript in the pathway that regulates gene expression. This long RNA molecule is the first product from a microRNA gene and serves as the precursor for functional molecules that influence protein production. Its existence is transient, as it is designed to be recognized and processed, initiating a cascade of events for cellular function.
Transcription and Structure of Pri-miRNA
The formation of a pri-miRNA begins in the cell’s nucleus, where miRNA genes are transcribed into a long RNA strand by the enzyme RNA Polymerase II. This is the same enzyme that creates messenger RNA (mRNA), integrating pri-miRNA production with the cell’s primary gene expression machinery. The resulting pri-miRNA transcript can be thousands of nucleotides in length.
Structurally, a pri-miRNA molecule shares features with mRNAs, including a protective 5′ cap and a poly-A tail that help stabilize it. Its defining characteristic is the presence of one or more hairpin-like structures, known as stem-loops. These structures form when the single-stranded RNA folds back on itself, with complementary base pairs creating a double-stranded stem and an unpaired loop at the end.
These hairpin formations are the specific features that cellular machinery will later recognize. The stem of the hairpin is a double-stranded RNA region, while the loop remains single-stranded. The junction between the single-stranded regions and the base of this hairpin stem creates a structural landmark that flags the pri-miRNA for processing.
Nuclear Processing by the Microprocessor Complex
Once transcribed, a pri-miRNA becomes the target of a molecular machine in the nucleus called the Microprocessor complex. This complex is composed of two main protein components: an enzyme named Drosha, which acts as molecular scissors, and a protein called DGCR8, which serves as a guide.
The DGCR8 protein identifies the pri-miRNA by recognizing the junction at the base of the hairpin stem. This ensures that only correct pri-miRNAs are processed. Once DGCR8 anchors the complex to the hairpin, it positions the Drosha enzyme for action.
Guided by DGCR8, Drosha makes a precise cut across the double-stranded RNA stem, approximately 11 nucleotides from the base of the structure. This cut liberates the hairpin from the long pri-miRNA transcript. The resulting molecule is a smaller, hairpin-shaped RNA of about 70 nucleotides, known as a precursor-miRNA, or pre-miRNA.
From Precursor to Mature miRNA
After processing, the new pre-miRNA is recognized by a transport protein called Exportin-5. This protein binds to the pre-miRNA and shuttles it out of the nucleus and into the cytoplasm through pores in the nuclear membrane. This export process also protects the pre-miRNA from being degraded within the nucleus.
Once in the cytoplasm, the pre-miRNA is processed by an enzyme called Dicer. Dicer recognizes the hairpin structure and makes a cut that removes the terminal loop. This results in a short, double-stranded RNA molecule approximately 22 nucleotides in length.
This double-stranded molecule is the miRNA duplex. One strand, the guide strand, is loaded into a protein complex called the RNA-induced silencing complex (RISC), while the other passenger strand is degraded. With the single-stranded mature miRNA, the RISC complex can find and regulate its target genes by binding to their messenger RNAs to silence them.
Consequences of Dysregulated Pri-miRNA Processing
Errors in the tightly controlled conversion of pri-miRNA to mature miRNA can have significant consequences. When pri-miRNA processing is dysregulated, it can disrupt gene regulation and is often linked to human diseases.
Mutations can occur within the sequence of a pri-miRNA itself. If these mutations alter the structure of the hairpin loop, they can prevent the DGCR8 protein from recognizing it. Without proper recognition, the Microprocessor complex cannot cleave the pri-miRNA, halting its production. This means the genes normally regulated by that miRNA are no longer controlled, which can contribute to disease.
Defects in the genes that produce Drosha and DGCR8 can also cause widespread issues. If these proteins are faulty, the processing of many different pri-miRNAs is impaired, leading to a reduction in mature miRNAs. For instance, disruption of the DGCR8 gene is a cause of DiGeorge syndrome. Faulty miRNA processing has also been linked to various cancers, where the loss of tumor-suppressing miRNAs can promote uncontrolled cell growth.