**11. miRNA-mediated mRNA decay**

Argonaute proteins prevent the formation of the closed-loop mRNA configuration by a murky mechanism that includes de-adenylation. MicroRNAs trigger de-adenylation and de-capping of the target mRNA. Proteins required for this process are components of the major deadenylase complex [CAF1, CCR4 and the NOT complex], the de-capping enzyme DCP2 and several de-capping activators. Thus, signal for translation is lost.

P-bodies or cytoplasmic processing bodies are nothing but ribosome-depleted areas inside a cell. Recent studies support the evidence that the mRNAs silenced by miRNAs are localized to P-bodies for storage or degradation. Induced by miRISC and RNA helicase activity, remodelled mRNPs may modify the translation initiation complex at the 5′ end of target mRNAs, which may cause translation repression and localization of mRNAs in P-bodies. Pbodies may facilitate the access of the de-capping factors to the cap structure, thus facilitating mRNA degradation. However, with the help of appropriate signals, stored mRNAs residing in P-bodies could be released and returned to the translational machinery of the cell [8, 28].

#### **12. miRNAs in different parts of the human brain**

**9. Post-initiation mechanisms**

142 Update on Amyotrophic Lateral Sclerosis

**10. Inhibition of translation initiation**

**11. miRNA-mediated mRNA decay**

several de-capping activators. Thus, signal for translation is lost.

[8].

Studies in the worm *C. elegans* and in mammalian cell cultures present evidences that miRNAs repress protein synthesis after translation is initiated. The contradictory observation that the targets of miRNAs appear to be actively translated while the corresponding protein product remains undetectable prompted the proposal that the nascent polypeptide chain might get degraded co-translationally. The identity of protease is unknown. Proteasome was excluded as a possibility because proteasome inhibitor does not restore the protein. Several other evidences lead to the suggestion that premature ribosome dissociation is caused by miRNAs

There was an observation by a group, where it was observed that the central domain of Argonaute proteins has sequence similarities to the cytoplasmic cap-binding protein *eIF4E* [eukaryotic translation initiation factor 4E], which is necessary for the cap-dependent transla‐ tion initiation in the cell. *eIF4E* binds to the m7Gppp-cap structure of mRNAs by stacking the methylated base of the cap between two tryptophans. At the equivalent position of the tryptophans in eIF4E, Argonaute proteins have phenylalanines that could mediate a similar interaction between the molecules. Consistently, Kiriakidou et al. [26] showed that human Argonaute 2 (AGO2) binds to m7GTP present on Sepharose beads. It was shown that substi‐ tuting one or both AGO2 phenylalanines with valine residues suspended the silencing activity. Using human cells it was shown that AGO2 associates with both *eIF6* and large ribosomal subunits. By binding to the large ribosomal subunit, *eIF6* prevents this subunit from joining with the small ribosomal subunit prematurely. *AGO2* recruits *eIF6*, thus stopping the associ‐

ation of the large and small ribosomal subunits, causing translation repression [8, 27].

Argonaute proteins prevent the formation of the closed-loop mRNA configuration by a murky mechanism that includes de-adenylation. MicroRNAs trigger de-adenylation and de-capping of the target mRNA. Proteins required for this process are components of the major deadenylase complex [CAF1, CCR4 and the NOT complex], the de-capping enzyme DCP2 and

P-bodies or cytoplasmic processing bodies are nothing but ribosome-depleted areas inside a cell. Recent studies support the evidence that the mRNAs silenced by miRNAs are localized to P-bodies for storage or degradation. Induced by miRISC and RNA helicase activity, remodelled mRNPs may modify the translation initiation complex at the 5′ end of target mRNAs, which may cause translation repression and localization of mRNAs in P-bodies. Pbodies may facilitate the access of the de-capping factors to the cap structure, thus facilitating One of the important processes is microRNA-mediated gene regulation, which is fine-tuning the expression of genes essential for the pathway. Using bioinformatics approach, one can easily understand that the cells of human brain are finely tuned by miRNAs. Prediction of miRNA target sites using bioinformatics approach can give us a whole bunch of gene names. But a major weakness of in silico analysis is the lack of experimental validation. For circum‐ venting such problem, a combinative approach can be implemented where target sites are predicted using multiple programmes. Out of miRNAs, which get expressed in the midbrain, cerebellum, hippocampus and frontal cortex, majority of them are from midbrain while almost equal proportions of microRNAs are expressed in the hippocampus and frontal cortex. The microRNAs in the cerebellum are less in number compared to miRNAs expressed in other regions considered. The target sites of the microRNA expressed in different parts of the brain had been predicted on the 35 genes related to ALS initially. The result obtained from miRanda [version 1.0] indicates that 477 target sites were predicted for mRNAs expressed in midbrain as compared to the target sites [411] predicted for miRNAs expressed in the hippocampus (**Figure 3**). However, total target sites predicted for miRNAs expressed in the cerebellum and frontal cortexes were 175 and 395, respectively (**Figure 3**). The number of targets for miRNAs expressed in cerebellum is considerably less comparing to binding sites for miRNAs expressed in other areas of the brain. Surprisingly, microRNA targets were distributed in the different regions of the genes and are not limited to only in 3′ UTR. Total 1456 target sites were predicted using miRanda for miRNAs considered in the study. Target sites in the 3′ untranslated region are comparatively less. A significant number of targets were also found in the 5′ UTR of the genes. Earlier, there was occurrence of miRNA target sites in coding region and 5′ UTR of genes was considered as exception in animals. But numerous recent evidences have established that microRNAs can target different regions of a gene and microRNA-based regulation is not confined only to 3′ UTR [9, 29]. Experiments aimed at identification of specific parameter or factor for effective targeting of 3′ UTR by miRNAs have failed considerably. Conversely, the existence of miRNA target sites in ORF of Nanog, *OCT4* and *SOX1*, during induction of stem cell pluripotency, reinforces the existence of miRNA-binding sites in other sites of the genes in animals. These results established the notion that microRNA-target sites are not only restricted to 3′ UTR in animals. Such variability in regions also adds cues for anticipating the mode of action especially their role in transcriptional, post-transcriptional and translation inhibition. As the majority of the algorithms developed for miRNA target site prediction consider only 3′ UTR, confirmation of 3′ UTR target site prediction using a combinatorial approach will be helpful. Target sites on ALS-related genes were classified according to the microRNAs expressed in four different regions of the brain (**Figure 3**). As there were less miRNAs expressed in cerebellum, this can explain small number of target sites predicted on genes associated to ALS. Analysis of miRNA targets in each gene associated with ALS showed

that there are no target sites that could be detected in 3′ UTR for mRNAs expressed in midbrain for *CASP1*, *GRIA1*, *GPX1*, *DAXX*, *CAT*, *SOD1*, *NEFM*, *NEFL* and *NEFH* genes. Furthermore 5′ UTR lack any target sites of same miRNAs in *CASP1*, *GRIA1*, *GPX1*, *DAXX*, *CAT*, *SOD1*, *NEFM*, *NEFL*, *NEFH*, *RAC1* and *TNF* genes. The miRNAs expressed in midbrain showed the least number of target sites in *RAC1* and *CASP1* genes, whereas 29 target sites were predicted in *NOS1*. Similar results were there for the miRNAs expressed in the cerebellum for the same genes. For instance, *RAC1* has on one target site whereas *NOS1* has 11 target sites. For miRNAs expressed in the hippocampus, *CASP1* and *GRIA1* genes have a few numbers of predicted sites [4] and *MAP3K5* was predicted to possess maximum target sites [30]. Similarly, *CASP1* and *SOD1* have two target sites each, while 23 target sites had been predicted for *TNFRSF1A* gene for miRNAs, expressed in the frontal cortex. On the other hand, target sites could not be predicted for six genes, namely, *GPX1*, *CYCS*, *CHP*, *CASP3*, *SOD1* and *RAB5A*, for miRNAs expressed in the cerebellum. No target sites could be predicted in 3′ UTR of *ALS2*, *BID*, *CASP1*, *GRIA1*, *DAXX*, *CCS*, *CAT*, *CASP9*, *TOMM40*, *TNF*, *SOD1*, *RAC1*, *NEFM* and *NEFL* genes. No target site could be predicted in *BCL2*, *CASP1*, *GRIA1*, *DAXX*, *CYCS*, *CASP3*, *TNF*, *RAC1*, *NEFM* and *NEFH* genes for miRNAs expressed in the cerebellum. These results demonstrate that the majority of the ALS genes lack any target site for miRNAs expressed in the cerebellum. miRNA targets were found in most of the genes, but surprisingly few genes, *CASP1*, *GRIA1*, *DAXX*, *CAT*, *SOD1* and *NEFM*, lack any miRNA target site in their 3′ UTR. This shows that complexity of regulations and numerous members of this play give the brain another level of entanglement [9] (**Figure 4**).

**Figure 3.** Percentage of miRNAs, expressed in the brain. From this diagram we can have an idea that the regulation of genes by miRNAs is widespread in various parts of the human brain (adapted from Ref. [9]).

**Figure 4.** Either dysregulation of miRNA biogenesis and function might result into ALS pathogenesis or ALS patho‐ genesis can cause dysregulation of miRNA biogenesis and function. Disrupted signalling at the neuromuscular junc‐ tion, caused by cytotoxicity associated with the faulty glutamate clearance or an overactive inflammatory response, results in neuromuscular degeneration. Dysregulation of key miRNAs triggers the altercations in cell physiology, re‐ sulting in ALS pathology (adapted from Ref. [84]).
