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Toward understanding post-transcriptional gene expression


04/23/2012
From purple petunias to the brain and memory; slicing and dicing our way toward understanding post-transcriptional gene expression...

Purple petunias  The brain

Imagine a better flower, a super purple, purple, superduper purple, petunia. That was the suggestion, the thought and the motivation for geneticist Rich Jorgensen to add an extra purple gene to a purple petunia back in 1986.

But what happened in Jorgensen's superduper purple petunia experiments? It failed! - but their final explanation has gone on to change our thinking into how genes are expressed and has provided us insights into whole new mechanisms of gene control and cellular regulation that may provide new keys for treatments of disease and new ways to engineer existing cells and entire organ systems' physiological health.

Jorgensen got white, pure white or spotted white flowers instead of purple ones: An unexpected result to say the least since he added extra purple producing genes. Numerous experiments later, it was determined that the RNA itself was interrupting its own expression through complementary binding and the formation of dsRNA molecules; the RNA it seems, was literally "interfering with itself", and the concept of RNA interference, or RNAi was born.

Later it was discovered that such RNA-RNA ds-binding activated a whole entire network of cellular immunity thought originally to be targeted at viral defense, and this lead to the discovery of a whole class of gene regulatory molecules that operate at the transcriptional level and seem to impact everything from flower color to mammalian memory and plasticity. Moreover it provides a wonderful insight into what the other 98% of the Transcriptome is doing: ~>2% of the Transcriptome is thought to code directly for proteins, with the other 98% being regulatory or functional RNA sequences like miRNA or siRNA. {Journal of Cell Science (2010) 123, 1819-1823, PLoS Biol 9(7): e1000625 (2011)}.

Today, Jorgensen's experiment and its subsequent discoveries are part of a growing arena of gene regulation which occurs post-transcriptionally. Post-transcriptional control of gene expression mediated by RNA interference via micro-RNA's (miRNA) and/or small interfering RNAs (siRNA) is revolutionizing, if not almost completely upending, much of what Science thought it knew about traditional gene regulation and expression. Micro-RNA's influence on gene expression has been shown to be important in an ever-increasing number of biological systems including:

Neuroscience: 
  1. Expression of microRNAs and their precursors in synaptic fractions of adult mouse forebrain (2008) J Neurochem. 106(2): 650-61
  2. Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner (2005) J Neurochem. 94: 896–905
Stem cell & development:
  1. MicroRNAs and Stem Cells: Control of Pluripotency, Reprogramming, and Lineage Commitment (2012) Circ Res. 110: 1014-1022
  2. MicroRNAs in Development and Disease (2011) Physiol. Rev. 91: 827-887
Autophagy & Cancer:
  1. MicroRNA-modulated autophagic signaling networks in cancer (2012) Inter. J Biochem. Cell Biol. 44(5): 733-736
T cell function:
  1. Dicer-dependent microRNA pathway safeguards regulatory T cell function (2008) JEM vol. 205(9): 1993-2004
  2. Dicer-dependent microRNA pathway controls invariant NKT cell development (2009) Journal of Immunology 183(4): 2506-2512
Cardiovascular biology & muscle:
  1. A New Level of Complexity: The Role of MicroRNAs in Cardiovascular Development (2012) Circ Res. 110: 1000-1013
  2. MicroRNAs Are Necessary for Vascular Smooth Muscle Growth, Differentiation, and Function" (2010) Arterioscler. Thromb. Vasc. Bio. 30: 1118-1126
Metabolic disease, metabolic syndrome & aging:
  1. Small but smart--microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing (2012) Cardiovasc Res. 93: 605-613
  2. MicroRNAs in Vascular and Metabolic Disease (2012) Circ Res. 110: 508-522
Endothelial function & apoptosis:
  1. Effects of down-regulation of microRNA-23a on TNF-{alpha}-induced endothelial cell apoptosis through caspase-dependent pathways (2012) Cardiovasc Res. 93(4): 623-32
Epigenetics:
  1. Non-coding RNAs as regulators of gene expression and epigenetics (2011) Cardiovasc Res. 90: 430-440
Found in a variety of higher organisms from plants to man, microRNAs or miRNAs are a growing family of small, non-protein coding regulatory genes that regulate the expression of homologous target-gene transcripts. They are products of at least two RNA processing stems, and the initial primary miRNA transcripts (called pri-mi-RNAs) are primarily transcribed by RNA polymerase II or III, and they are classical stem-loop structures of greater than 70 nucleotides in length.

These pri-miRNA transcripts are then processed within the cell nucleus by a multiprotein complex called the "microprocessor" and cleaved at the stem of the hairpin loop to generate a shorter RNA structure of ~ 70 nucleotides in length called the precursor - or pre-miRNA.

The processed pre-miRNA are then exported into the cytoplasm and loaded onto another multi-protein complex processing machine which contains a ribonuclease III containing protein called Dicer, proteins from the Argonaute protein family (Agos, a.k.a eIF2Cs), and other accessory cellular factors particular to each system. Each protein has a specific function within the RISC complex. Dicer will mediate the cleavage of the pre-miRNA to form the mature miRNAs of approximately 22 nucleotides in length. These will then be bound by specific Argonaute proteins to form together with other cellular factors, the effector silencing complex or RISC which is able to modulate protein expression upon binding by sequence complementarity to the specific region of 3' untranslated region (UTR) of specific mRNAs to alter their protein synthesis – and thus their expression within the cell.



Dicer pre-miRNA process
Figure 2A

As described, Dicer is a multidomain RNase III like enzyme involved in the initial steps of RNA interference (RNAi) and microRNA (miRNA) pathways. Dicer has been shown to be necessary and sufficient to cut long dsRNA and miRNA precursors into small (21-25 nt) RNAs. In animals, the small RNA products of Dicer are further incorporated into a multiprotein RNA induced silencing complex (RISC), which target mRNAs in a sequence specific manner to induce mRNA cleavage (guided by siRNAs) or inhibition of translation (guided by miRNAs).

Argonaute proteins are the catalytic components of the RNA-induced silencing complex (RISC), {Cenik ES, Zamore PD. (2011). "Argonaute proteins" Current Biology 21 (12): R446–9.} Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) {Ghildiyal M, Zamore PD (2009). "Small silencing RNAs: an expanding universe". Nat. Rev. Genet 10 (2): 94–108}.

Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity, which typically leads to silencing of the target. Argonaute proteins are named after the argonaute (AGO) phenotype of Arabidopsis thaliana mutants, which itself was named after its resemblance to Argonauts, a family of a kind of octopus in the genus Argonauta—so named after the vessel Argo, which in Greek mythology was a vessel on a dangerous but rewarding mission to retrieve the Golden Fleece. Interestingly too, we've all seen Argonaute proteins before in biochemistry. They are more commonly referred to as Eukaryotic translation initiation factor 2C (eIF2C) proteins. There are four isoforms, eIF2C1-4, with eIF2C1-3 sharing identical C-terminal sequences.

Ago2 is the only member of the family with endonuclease activity and thus it has achieved the most scientific attention. Ago2 is identical to eIF2c; its alternative name is eIf-2C2. The protein is required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC). The 'minimal RISC' appears to include EIF2C2/AGO2 bound to a short guide RNA such as a microRNA (miRNA) or short interfering RNA (siRNA). These guide RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by EIF2C2/AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. The inhibition of translational initiation leads to the accumulation of the affected mRNA in cytoplasmic processing bodies (P-bodies), where mRNA degradation subsequently occurs. See Figure 2A above. Recent evidence also demonstrates that Ago2 alone, without binding to Dicer can also directly process pre-miRNA (Figure 2B below) {adapted from Cell Research (2010) 20:735-737}.


RISC
Figure 2B

In addition to gene-silencing, EIF2c also can act as an activator of gene expression. For example with eIF2C, it binds to the AU element of the 3'-UTR of the TNF (TNF-alpha) mRNA and up-regulates translation under conditions of serum starvation. Moreover, binding of eIF2C may also be required for direct transcriptional activation of transcriptional gene silencing (TGS) elements, in which short RNAs known as antigene RNAs or agRNAs are transcriped and function in the direct the transcriptional repression of complementary promoter regions of specific genes.

Ago proteins localize to the cytoplasm of somatic cells and are concentrated in cytoplasmic processing bodies (P-bodies). {Genome Biology 2008, 9:210}

Biosensis is pleased to offer excellent new polyclonal antibodies to two key players in the RNA interference and/or micro-RNA gene regulation pathways: A chicken polyclonal anti-Dicer antibody specific for rodent Dicer (Catalog number: C-1557-100) and a pan, anti-mouse eIF2c (Argonaute protein 2) antibody capable of staining Ago2 proteins and its isoforms (Ago 1-3) (Catalog number: R-1556-100).

Rabbit polyclonal antibody to mouse elF2c protein: Affinity purified Biosensis' antibody against eIF2c protein (Catalog Number: R-1556-100) has been used to study the role of small RNA pathways in synaptic functions, particularly in plasticity and learning. Anti-eIF2c antibody has been used to demonstrate that a diverse population of microRNAs is expressed within dendrites and within dendritic spines. Mature microRNAs, which silence target mRNAs via incorporation into the RNA-induced silencing complex (RISC), are known to bind to the Argonaute homologue eIF2c as part of the RISC complex. {Journal of Neurochemistry, 2005, 94, 896–905; Journal of Neurochemistry , 2008,106 , 650–661}.

Chicken polyclonal antibody to mouse Dicer protein: Affinity purified Biosensis' anti-Dicer chicken polyclonal antibody (Catalog Number: C-1557-100) recognizes two major bands at 220 kDa (which co-migrated with recombinant dicer) and a smaller band at 125 kDa. Dicer appears to undergo some fragmentation during processing accounting for the lower molecular weight fragment. The antibody has been shown specific for mouse Dicer in adult mouse brain extracts by western blot, immunoprecipitation, and siRNA experiments. In addition only anti-dicer immune preceipitated dicer protein properly bound a synthetic dsRNA construct but not poly c beads. {Journal of Neurochemistry, 2005, 94, 896–905}

Dicer is expressed in many brain regions, in a punctate pattern in the somatodendritic compartment of large neurons, some interneurons and endothelial cells. Neuropil was prominently labeled but only light staining was evident in astrocytes, oligodendrocytes and white matter. Electron microscopy immunocytochemistry using peroxidase-based detection confirmed that dicer-like immunoreactivity was present within dendritic spines and appeared to be particularly associated with PSDs.{ Journal of Neurochemistry, 2005, 94, 896–905}.

Together these new reagents provide new and useful reagents for the examination in rodents of miRNA processing and influence in a variety of cell and organ systems including the brain and the study of such wonderful scientific arenas as memory and dendritic plasticity.
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