There are four major types of introns. Group I introns, found in some rrna genes, are self-splicing: they can catalyze their own removal.

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3 Continuous genes - Intron: Many eukaryotic genes contain coding regions called exons and noncoding regions called intervening sequences or introns. The average human gene contains from eight to nine introns. All the introns and the exons are initially transcribed into RNA but, after transcription, the introns are removed by splicing and the exons are joined to yield the mature RNA. Introns have now been observed in archaea, bacteriophages, and even some eubacteria. Introns are present in mitochondrial and chloroplast genes as well as the nuclear genes of eukaryotes. In eukaryotic genomes (All classes of eukaryotic genes contain intron), the size and number of introns appear to be directly related to increasing organismal complexity. Yeast genes contain only a few short introns. The number and size of introns vary widely: some eukaryotic genes have no introns, whereas others may have more than 60; intron length varies from fewer than 200 nucleotides to more than 50,000. Introns tend to be longer than exons, and most eukaryotic genes contain more noncoding nucleotides than coding nucleotides. 3

4 There are four major types of introns. Group I introns, found in some rrna genes, are self-splicing: they can catalyze their own removal. Group II introns in some protein-encoding genes of mitochondria, chloroplasts; they also are self-splicing, but their splicing differs from that of the group I introns. Nuclear pre-mrna introns are the best studied; they include introns located in the protein-encoding genes of the eukaryotic nucleus. The splicing mechanism by which these introns are removed is similar to that of the group II introns, but nuclear introns are not self-splicing; their removal requires snrnas and a number of proteins. Transfer RNA introns, found in trna genes, utilize yet another splicing mechanism that relies on enzymes to cut and reseal the RNA. 4

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6 In eukaryotic cells, ribosomes bind to a modified 5 end of mrna, as discussed later in the chapter. 6

7 In bacterial cells, transcription and translation take place simultaneously; while the 3 end of an mrna is undergoing transcription, ribosomes attach to the Shine Dalgarno sequence near the 5 end and begin translation. eukaryotic mrna is extensively altered after transcription. Changes are made to the 5 end, the 3 end, and the protein coding section of the RNA molecule. 7

8 The cap consists of an extra nucleotide (G) at the 5 end of the mrna and methyl groups (CH3) on the base in the newly added nucleotide and on the 2 -OH group of the sugar of one or more nucleotides at the 5 end. In cytoplasm, Cap-binding proteins recognize the cap and attach to it; a ribosome then binds to these proteins and moves downstream along the mrna until the start codon is reached and translation begins. The 5 end of pre-mrna can be represented as 5 pppnpnpn This guanine nucleotide is attached to the premrna by a unique 5 5 bond, which is quite different from the usual 5 3 phosphodiester bond RNA molecules transcribed by polymerases I and III (rrnas, trnas, and some snrnas) are not capped. 8

9 The stability conferred by the poly(a) tail depends on the proteins that attach to the tail and on its length. The poly(a) tail also facilitates attachment of the ribosome to the mrna. 50 to 250 or adenine at the 3 end is added, forming a poly(a) tail (polyadenylation). Many eukaryotic genes transcribed by RNA polymerase II are transcribed beyond the end of the coding sequence; most of the extra material at the 3 end is then cleaved and the poly(a) tail is added. Processing of the 3 end of pre-mrna requires sequences both upstream and downstream of the cleavage site. The consensus sequence AAUAAA is usually from 11 to 30 nucleotides upstream of the cleavage site and determines the point at which cleavage will take place. A sequence rich in uracil nucleotides (or in guanine and uracil nucleotides) is typically downstream of the cleavage site. Poly(U) tails are added to the 3 ends of some mrnas, micrornas, and small nuclear RNAs. evidence suggests that poly(u) tails may facilitate their degradation. 9

10 Splicing requires three sequences in the intron splice site, 2. 3 splice site; Most introns in premrnas begin with GU and end with AG, indicating that these sequences play a crucial role in splicing. Indeed, changing a single nucleotide at either of these sites prevents splicing. 3. the branch point, which is an adenine nucleotide that lies upstream of the 3 splice site The result is that the 5 phosphate group of the G nucleotide is now attached to the 2 -OH group of the A nucleotide at the branch point 10

11 A key feature of the process is a series of interactions between the mrna and the snrnas and between different snrnas. These interactions depend on complementary base pairing between the different RNA molecules and bring the essential components of the pre-mrna transcript and the spliceosome close together, which make splicing possible. Immediately after splicing, a group of proteins called the exon-junction complex (EJC) is deposited approximately 20 nucleotides upstream of each exon exon junction on the mrna. The EJC promotes the export of the mrna from the nucleus into the cytoplasm. 11

12 The splicing of group II introns is similar to the spliceosomal-mediated splicing of nuclear genes, and splicing generates a lariat structure. Because of these similarities, group II introns and nuclear pre-mrna introns have been suggested to be evolutionarily related; perhaps the nuclear introns evolved from self-splicing group II introns and later adopted the proteins and snrnas of the spliceosome to carry out the splicing reaction. 12

13 Alternative processing of pre-mrnas is common in multicellular eukaryotes. For example, researchers estimate that more than 90% of all human genes undergo alternative splicing. 13

14 A variety of mechanisms can change in RNA. In some cases, molecules called guide RNAs play a crucial role. A grna contains sequences that are partly complementary to segments of the RNA, and the two molecules undergo base pairing in these sequences. After the mrna is anchored to the grna, the mrna undergoes cleavage and nucleotides are added, deleted, or altered according to the grna. In other cases, enzymes bring about base conversion. For example In some cases, an enzyme deaminates a cytosine base, converting it into uracil. This conversion changes a codon that specifies an amino acid into a stop codon that prematurely terminates translation, resulting in the shortened protein. 14

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17 trnas genes may be in clusters or scattered about the genome. In E. coli, the genes for some trnas are present in a single copy, whereas the genes for other trnas are present in several copies; eukaryotic cells usually have many copies of each trna gene. In E. coli, (and usually in Eukaryotic) several trnas are usually transcribed together as one large precursor trna, which is then cut up into pieces, each containing a single trna. Additional nucleotides may then be removed one at a time from the 5 and 3 ends of the trna in a process known as trimming. Some eukaryotic and archeal trna genes possess introns of variable length that must be removed in processing. For example, about 40 of the 400 trna genes in yeast contain a single intron that is always found adjacent to the 3 side of the anticodon. The trna introns is small and do not have the consensus sequences found at the intron exon junctions of premrnas. The splicing process for trna genes is quite different from the spliceosome-mediated reactions. 17

18 End All the modified bases in trnas are produced by the chemical alteration of the four standard RNA bases after transcription. 18

19 1 Ribosomes typically contain about 80% of the total cellular RNA. 19

20 1 In E. coli, the 30S precursor is methylated in several places, and then cleaved and trimmed to produce 16S rrna, 23S rrna, and 5S rrna, along with one or more trnas. 2 Eukaryotic rrnas undergo similar processing. Small nucleolar RNAs (snornas) help to cleave and modify eukaryotic rrnas and assemble the processed rrnas into mature ribosomes. snornas associate with proteins to form ribonucleoprotein particles (snornps). The snornas have extensive complementarity to the rrna sequences in which modification takes place. 20

21 These double-stranded RNA molecules are chopped up by an enzyme appropriately called Dicer, resulting in tiny RNA molecules that are unwound to produce sirnas and mirnas 21

22 However, RNA interference is also responsible for regulating a number of key genetic and developmental processes, including changes in chromatin structure, translation, cell fate and proliferation, and cell death. Geneticists also use RNAi for blocking the expression of specific genes. Usually, sirnas have exact complementarity with their target mrna or DNA sequences and suppress gene expression by degrading mrna or inhibiting transcription, whereas mirnas often have limited complementarity with their target mrnas and often suppress gene expression by inhibiting translation. Finally, mirnas usually silence genes that are distinct from those from which the mirnas were transcribed, whereas sirnas typically silence the genes from which the sirnas were transcribed. The genes that encode mirnas are transcribed into longer precursors, called primary mirna (pri-mirna), that range from several hundred to several thousand nucleotides in length. The pri-mirna is then cleaved into one or more smaller RNA molecules with a hairpin. Dicer binds to this hairpin structure and removes the terminal loop. One of the mirna strands is incorporated into the RISC; the other strand is released and degraded. The RISC attaches to a complementary 22

23 sequence on the mrna, usually in the 3 untranslated region of the mrna. The region of close complementarity, called the seed region, is quite short, usually only about seven nucleotides long. Because the seed sequence is so short, each mirna can potentially pair with sequences on hundreds of different mrnas. Furthermore, a single mrna molecule may possess multiple mirna-binding sites. The inhibition of translation may require binding by several RISC complexes to the same mrna molecule. 22

24 Humans have more than 450 distinct mirnas; more than one-third of all human genes are regulated by mirnas. Most mirna genes are found in regions of noncoding DNA or within the introns of other genes. 23

25 Less is known about Piwi-interacting RNAs (pirnas). They are somewhat longer than sirnas and mirnas and are derived from long, single-stranded RNA transcripts, in contrast with sirnas and mirnas, which are processed from double-stranded RNA. Piwi-interacting RNAs combine with Piwi proteins, which are related to Argonaute, and inhibit the expression of transposons, primarily in the germ cells of animals. Although the mechanism of transposon silencing by pirnas is not well understood, it appears to entail degradation of mrna transcribed from transposons and changes in chromatin structure that inhibit the transcription of transposons. 24