1. Regulation of small RNAs
Jessica Jaraba Wallace
Genomics
Advanced Genetic Master
December, 2017

2. Conclusions Introduction Objectives Materials and methods Bibliography
Ribosomal RNA (<15%)
1.5 – 2 %
Transfer RNA (>80%)
Small nuclear RNA
Long non-coding RNA (>200nt)
For decades, one of the long-standing principles of molecular biology was that DNA acts as a template for transcription of messenger RNAs, which served as an intermediate molecule to transfer the genetic information to proteins translation. The most well-studied sequences in the human genome are those of protein-coding genes. However, a rapidly growing number of exceptions to this rule have been reported over the past decades, the coding exons of these genes account for only 1,5% of the genome, a proportion that increases to 2% if untranslated regions (UTRs) are considered. On the other hand, the genome encodes tens of thousands of small and long RNA transcripts that do not code for proteins which have crucial functional importance for normal development and physiology and for disease.
They include long known classes of RNAs involved in translation such as transfer RNAs and ribosomal RNAs. Small nuclear RNAs involved in splicing events.
More recently, a growing number of new regulatory small short regulatory non-coding RNAs (of less than 200 nucleotides of length) have been discovered, which act as key regulators of gene expression in many different cellular pathways and systems. Additionally, the human genome encodes several thousand long non-protein coding RNAs of more than 2000 nucleotides in length, some of which play crucial roles in a variety of biological processes such as epigenetic control of chromatin, promoter-specific gene regulation, mRNA stability, X-chromosome inactivation and imprinting.
Short non-coding RNA (<200nt)
Other?

3. Short non-coding RNA (<200nt)
Introduction
Introduction
Small ncRNAs
Small ncRNAs
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
Classification
miRNA ≈22 bp
piRNA bp
siRNA
Short non-coding RNA (<200nt)

4. Short non-coding RNA (<200nt)
Introduction
Introduction
Small ncRNAs
Small ncRNAs
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
Classification
miRNA ≈22 bp
piRNA bp
siRNA
Most widely studied class of ncRNA, about 22nt.
They repress the expression of their target sites.
Short non-coding RNA (<200nt)

5. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
miRNA Biosynthesis
The most widely studied class of ncRNAs are miRNAs, which are small ncRNAs of about 22 nucleotides that directly interact with partially complementary target sites and repress their expression.
In general, mammalian miRNAs are genomically encoded and transcribed by RNA Polymerase II as primary miRNA trasncriprs (pri-miRNAs): usually several thousand nucleotides long, which get processed by the microprocessor complex consisting of the RNAse II enzyme Drosha and the pri-miRNA binding protein DGCR8: cuts the 5' and 3' ends of the pri-miRNA. Drosha may also form larger complexes with other proteins to regulate the processing of specific pri-miRNAs. The resulting pre-miRNA (giving a short hairpin of 60–70 nucleotides long ) gets translocated to the cytosol by the energy-dependent mechanism involving exportin5, where it is further processed into an approximately 21 nt long (without the terminal loop) dsRNA by Dicer and other accessory proteins, including the transactivation response RNA binding protein (TRBP), the protein activator of the dsRNA-dependent protein kinase (PACT). Subsequently, this dsRNA gets incorporated into the RNA-induced silencing complex (RISC) which includes the mentioned proteins and the Ago proteins, which only keeps one strand of the mature mRNA (different hypothesis explain which one it keeps).
- This one single-stranded (name guide) of the duplex (which is complementary to the target mRNA). The activated RISC can bind the target mRNA, and direct its degradation or repress its translation. However, it has been reported that in some cases, miRNAs can also up-regulate the expression of their targets.
- While the other strand of the duplex (named passenger or miRNA*) is usually degraded
(Tomaselli et al. 2014)

6. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
miRNA Gene Regulation
Regulate the translation of more than 60% of the genes:
Proliferation, differentiation, apoptosis and development.
Specific individual targets.
Regulators of a process; hundreds of genes simultaneously and many types of miRNA regulate cooperatively.
Guide mRNAs directly interacts with partially complementary target sites located in the 3’ UTR of the target mRNAs and repress their expression by two main means:
- The inhibition of translation initiation.
- mRNA degradation initiated by deadenylation and decapping, making the mRNAs accessible for exoribonucleases.
miRNAs are estimated to regulate the translation of more than 60% of protein-coding genes. They are involved in regulating many processes, including proliferation, differentiation, apoptosis and development. Whereas some miRNAs regulate specific individual targets, other can function as master regulators of a process, so key miRNAs regulate the expression levels of hundreds of genes simultaneously, and many types of miRNAs regulate their targets cooperatively.
(Yu et al. 2016)

7. Short non-coding RNA (<200nt)
Introduction
Introduction
Small ncRNAs
Small ncRNAs
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
Classification
miRNA ≈22 bp
piRNA bp
siRNA
Most recently identified classes, about 24-30nt.
Dicer-Independent; incorporated into the PIWI subfamily of ARGO proteins.
Transcribed from regions that contain active transposable elements, other repetitive elements and piRNA clusters.
Short non-coding RNA (<200nt)

8. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
piRNA Biosynthesis
Primary processing
Ping-pong amplification cycle (secondary processing)
Predominant in somatic follicle cells (also in germline cells)
Predominant in germline cells
piRNA biogenesis occurs by two pathways:
Primary processing: which occurs predominantly in somatic follicle cells but also in germline cells.
Secondary processing: which occurs via a feed forward loop called ping-pong amplification cycle, occurs only in germline cells.
The primary processing pathway derives from long single-stranded antisense precursors RNA that after processing, are loaded into PIWI.
The ping-pong amplification pathway:
- Either PIWI or Aub and its associated antisense piRNA cleaves the transposon transcripts and sense piRNA precursors produced from piRNA clusters. The resulting products are then loaded onto Ago3 were its processing fishes to a mature sense piRNA.
- Ago3 and its associtated mature sense piRNA further cleave the target antisense piRNA precursors based on the sequence complementarity. The resulting antisense piRNA intermediates are loaded onto Aub, where it is processed into the mature antisense piRNA.
This sense and antisense piRNA come from: the transposon transcripts and the piRNA clusters, as already mentioned. But can also be initiated by the primary processing pathway. piRNAs can also be transmitted vertically through maternal inheritance, thereby providing a consistent defence against retrotransposons.
(Patil et al. 2017)

9. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
piRNA Regulation
1) Transposon silencing
2) Epigenetic regulation: DNA methylation
Upon biogenesis piRNAs associated to PIWI proteins. Thanks to piRNA complementarity:
- Control the expression and mobilization of sense transposon-derived transcripts:
- Transcriptional silencing: by forming heterochromatic structures at the transposon loci.
- By transcript destruction by the slicer activity of the PIWI proteins.
- Or by translational inhibition.
- They are also linked to the DNA methylation during epigenetic regulation. Consistently with this a single piRNA was recently reported to mediate locus-specific methylation of an imprinted region.
A single piRNA was recently reported to mediate locus-specific methylation of an imprinted region.
(Khurana and Theurkauf 2010)

10. Short non-coding RNA (<200nt)
Introduction
Introduction
Small ncRNAs
Small ncRNAs
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
Classification
miRNA ≈22 bp
piRNA bp
siRNA
Short non-coding RNA (<200nt)
Derived from double-stranded precursors RNAs

11. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
siRNA Biosynthesis
Endogenous
Exogenous
(viral dsRNA)
Endogenous siRNAs precursors are derived from repetitive sequences, sense-antisense pairs or long stem-loop structures.
Exogenous small interfering RNAs are derived from double-stranded genomic replication intermediates of invading RNA viruses.
Small interfering RNAs are transferred to the cytoplasm and are cleaved into 20-25nt by Dicer complex. It is then loaded into the Ago proteins, where one strand is retained in the RISC complex (the guide strand), while the “passenger” strand is discarded.
Discard the “passenger” strand

12. Introduction Small ncRNAs Conclusions Bibliography Endogenous
siRNA Regulation
Endogenous
Exogenous
(viral dsRNA)
Regulation of a small group of mRNAs
Endo-siRNAs contribute to maintenance of genomic stability in the female germline.
Antiviral defence mechanism: the siRNA further targets viral replication intermediates and viral genomic RNA.
This siRNA have different regulation:
The endogenous siRNA:
The siRNA directs the RISC complex to regulate a small group of mRNAs.
While most of the transposons are processed by piRNA, those from the female germline gice rise to dsRNAs, which can be processed into endo-siRNAs. So they contribute to the maintenance of genomic stability in the female germline
Exo-siRNAs are a key in the antiviral defence mechanism as the siRNA further targets viral replication intermediates and viral genomic RNA.

13. Introduction Small ncRNAs Conclusions Bibliography siRNA Regulation
The ways they control this is by:
Recognizing complementary mRNA and degrade it. After cleavage, functional siRISC is regenerated.
They can also silence targets by translational repression.
Finally, it can also direct chromatin modifications.
(Carthew and Sontheimer 2009)

14. Conclusions Introduction Small ncRNAs Conclusions Bibliography
Materials and methods
Conclusions
Conclusions
Bibliography
Bibliography
Most RNAs are non-coding RNAs, which don’t translate into proteins, but still have important functions.
miRNAs regulate the translation of more than 60% of the genes.
piRNAs mostly target transposons transcripts in germline cells. However, also mediate DNA methylation.
- siRNA regulate a small group of mRNAs.
- Endo-siRNA contribute to the maintenance of genomic stability in the female line.
- Exo-siRNA act as an antiviral defence mechanism.
Both miRNA and siRNA biogenesis are mediated by DICER, yet piRNA are DICER-independent.
piRNA biogenesis occurs by two pathways:
Primary processing: which occurs predominantly in somatic follicle cells but also in germline cells.
Secondary processing: which occurs via a feed forward loop called ping-pong amplification cycle, occurs only in germline cells.
The primary processing pathway derives from long single-stranded antisense precursors RNA that after processing, are loaded into PIWI.
The ping-pong amplification pathway:
- Either PIWI or Aub and its associated antisense piRNA cleaves the transposon transcripts and sense piRNA precursors produced from piRNA clusters. The resulting products are then loaded onto Ago3 were its processing fishes to a mature sense piRNA.
- Ago3 and its associtated mature sense piRNA further cleave the target antisense piRNA precursors based on the sequence complementarity. The resulting antisense piRNA intermediates are loaded onto Aub, where it is processed into the mature antisense piRNA.
This sense and antisense piRNA come from: the transposon transcripts and the piRNA clusters, as already mentioned. But can also be initiated by the primary processing pathway. piRNAs can also be transmitted vertically through maternal inheritance, thereby providing a consistent defence against retrotransposons.

15. Introduction Small ncRNAs Conclusions Bibliography
Carthew RW, Sontheimer EJ (2009) Review Origins and Mechanisms of miRNAs and siRNAs. Cell 136:642–655. doi: /j.cell
Hombach S, Kretz M (2016) Biology and Functioning. doi: /
Khurana JS, Theurkauf W (2010) piRNA, transposon silencing, and Drosohila germline development. 191:905–913. doi: /jcb
Ku H, Lin H (2017) PIWI proteins and their interactors in piRNA biogenesis, germline development and gene expression. 205–218. doi: /nsr/nwu014
Mehta P, Goyal S, Wingreen NS (2008) A quantitative comparison of sRNA-based and protein-based gene regulation. doi: /msb
Patil VS, Zhou R, Rana TM (2017) Gene regulation by non-coding RNAs. doi: /
Rna N, Esteller M (2011) Non-coding RNAs in human disease. Nat Publ Gr 12:861–874. doi: /nrg3074
Shimoni Y, Friedlander G, Hetzroni G, et al (2007) Regulation of gene expression by small non-coding RNAs : a quantitative view. 1–9. doi: /msb
Tomaselli S, Bonamassa B, Alisi A, et al (2014) ADAR enzyme and miRNA story : A nucleotide that can make the difference ADAR Enzyme and miRNA Story : A Nucleotide that Can Make the Difference. doi: /ijms
Yu A, Tian Y, Tu M, et al (2016) Minireview MicroRNA Pharmacoepigenetics : Posttranscriptional Regulation Mechanisms behind Variable Drug Disposition and Strategy to Develop More Effective Therapy. 308–319.
piRNA biogenesis occurs by two pathways:
Primary processing: which occurs predominantly in somatic follicle cells but also in germline cells.
Secondary processing: which occurs via a feed forward loop called ping-pong amplification cycle, occurs only in germline cells.
The primary processing pathway derives from long single-stranded antisense precursors RNA that after processing, are loaded into PIWI.
The ping-pong amplification pathway:
- Either PIWI or Aub and its associated antisense piRNA cleaves the transposon transcripts and sense piRNA precursors produced from piRNA clusters. The resulting products are then loaded onto Ago3 were its processing fishes to a mature sense piRNA.
- Ago3 and its associtated mature sense piRNA further cleave the target antisense piRNA precursors based on the sequence complementarity. The resulting antisense piRNA intermediates are loaded onto Aub, where it is processed into the mature antisense piRNA.
This sense and antisense piRNA come from: the transposon transcripts and the piRNA clusters, as already mentioned. But can also be initiated by the primary processing pathway. piRNAs can also be transmitted vertically through maternal inheritance, thereby providing a consistent defence against retrotransposons.