Ch.8 Eukaryotic transcription Eukaryotic transcription is more complex than prokaryotic transcription and, until recently, it has seemed that every eukaryotic.

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1 ch.8 Eukaryotic transcription Eukaryotic transcription is more complex than prokaryotic transcription and, until recently, it has seemed that every eukaryotic gene was unique requiring its own transcription machinery. Eukaryotic transcription is more complex than prokaryotic transcription and, until recently, it has seemed that every eukaryotic gene was unique requiring its own transcription machinery.

2 1. INTRODUCTION

3 heterogeneous nuclear RNA :The original RNA transcripts found in eukaryotic nuclei before post-transcriptional modifications. A diverse assortment of RNA types found in the nucleus, including mRNA precursors (pre-mRNA) and other types of RNA. Abbreviated hnRNA. heterogeneous nuclear RNA :The original RNA transcripts found in eukaryotic nuclei before post-transcriptional modifications. A diverse assortment of RNA types found in the nucleus, including mRNA precursors (pre-mRNA) and other types of RNA. Abbreviated hnRNA. http://www.everythingbio.com/glos/definition.php?word=heterogeneous+nuclear+ RNA http://www.everythingbio.com/glos/definition.php?word=heterogeneous+nuclear+ RNA Small nuclear RNA (snRNA) Small nuclear RNA (snRNA) A class of small RNA molecules that are found within the nucleus of eukaryotic cells. They are transcribed by RNA polymerase II or RNA polymerase III and are involved in a variety of important processes such as RNA splicing (removal of introns from hnRNA), regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA), and maintaining the telomeres. They are always associated with specific proteins, and the complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. These elements are rich in uridine content. A class of small RNA molecules that are found within the nucleus of eukaryotic cells. They are transcribed by RNA polymerase II or RNA polymerase III and are involved in a variety of important processes such as RNA splicing (removal of introns from hnRNA), regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA), and maintaining the telomeres. They are always associated with specific proteins, and the complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. These elements are rich in uridine content. http://en.wikipedia.org/wiki http://en.wikipedia.org/wiki

4 Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that guide chemical modifications (methylation or pseudouridylation) of ribosomal RNAs (rRNAs) and other RNAs (tRNAs and other small nuclear RNAs (snRNAs)). They are classified under snRNA. snoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs (gRNA) that direct RNA editing in trypanosomes. are a class of small RNA molecules that guide chemical modifications (methylation or pseudouridylation) of ribosomal RNAs (rRNAs) and other RNAs (tRNAs and other small nuclear RNAs (snRNAs)). They are classified under snRNA. snoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs (gRNA) that direct RNA editing in trypanosomes.

5 scRNA(small cytoplasmic RNA) scRNA 参与蛋白质的合成和运输, 如 SRP 颗粒就是一种由一个 7SRNA 和蛋 白质组成的核糖核蛋白体颗粒, 主要功 能是识别信号肽, 并将核糖体引导到内 质网。 scRNA 参与蛋白质的合成和运输, 如 SRP 颗粒就是一种由一个 7SRNA 和蛋 白质组成的核糖核蛋白体颗粒, 主要功 能是识别信号肽, 并将核糖体引导到内 质网。

6 microRNAs (miRNA) microRNAs (miRNA) are single- stranded RNA molecules of about 21 – 23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down- regulate gene expression. microRNAs (miRNA) are single- stranded RNA molecules of about 21 – 23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed but miRNAs are not translated into protein (non-coding RNA); instead each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA and finally into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down- regulate gene expression. The stem-loop secondary structure of a pre-microRNA from Brassica oleracea.

7 Catalytic RNA- Catalytic RNA- enzymatically active RNA molecules -Ribozyme From Wikipedia, the free encyclopedia

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9 There are three distinct RNA polymerases in a eukaryotic cell nucleus which define the three major classes of eukaryotic transcription unit. Each type of RNA polymerase is a complex of many polypeptide subunits. Eukaryotic RNA polymerases cannot find or bind to a promoter by themselves. Each requires the binding of assembly factors and a positional factor to locate the promoter and to orient the polymerase correctly. 2. RNA polymerases

10 All genes that are transcribed and expressed via mRNA are transcribed by RNA polymerase II. RNA polymerase II.

11 A significant difference in the transcription of eukaryotic and prokaryotic mRNAs is that initiation at a eukaryotic promoter involves a large number of factors that bind to a variety of cis-acting elements. A significant difference in the transcription of eukaryotic and prokaryotic mRNAs is that initiation at a eukaryotic promoter involves a large number of factors that bind to a variety of cis-acting elements. Transcription factor: Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. Transcription factor: Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor.

12 3. promoters 3.1 core promoter

13 Bacterial Promoters: Pol II Promoters: NOT a TATA box

14 TATA box(Hogness box )Eukaryotic promoter region analogous to the Pribnow box. It is a conserved A/T rich septamer found about 25-30 bp upstream of a gene,which is the target site of eukaryotic RNA polymerase II. TATA box(Hogness box )Eukaryotic promoter region analogous to the Pribnow box. It is a conserved A/T rich septamer found about 25-30 bp upstream of a gene,which is the target site of eukaryotic RNA polymerase II. 是控制转录精确性的序列, 决定转录的方向和精 确的转录起始点。 是控制转录精确性的序列, 决定转录的方向和精 确的转录起始点。 Consensus sequence: T 82 A 97 T 93 A 85 A 63 (T 37 )A 83 A 50 (T 37 ). Consensus sequence: T 82 A 97 T 93 A 85 A 63 (T 37 )A 83 A 50 (T 37 ).

15  initiator=INR 一些真核基因的上游无典型的 TATA 盒，但有起始子元件 INR. 也有 一些真核基因既有 TATA 盒，也有 INR 序列。 一些真核基因的上游无典型的 TATA 盒，但有起始子元件 INR. 也有 一些真核基因既有 TATA 盒，也有 INR 序列。

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17 3.2 upstream promoter element =UPE

18 CAAT 盒 最常见的为转录起始点上游 - 75bp 的 CAAT 盒, 一致序列为 GGCC/TCAATCT, 因其保守序列为 CAAT 而得名。 与 start point 距离变化较大仍不 影响功能, 可以以不同的方向起作用。 突变敏感性试验表明其在决定启 动子的强弱（或效率）上起很大作用。

19 GC box GC box GGGCGG, – 90bp 左右, 在不同启动子 中位置可有变化，在一个启动子中可有多 个拷贝, 改变方向不影响功能. GGGCGG, – 90bp 左右, 在不同启动子 中位置可有变化，在一个启动子中可有多 个拷贝, 改变方向不影响功能.

20 A significant difference in the transcription of eukaryotic and prokaryotic mRNAs is that initiation at a eukaryotic promoter involves a large number of factors that bind to a variety of cis-acting elements. A significant difference in the transcription of eukaryotic and prokaryotic mRNAs is that initiation at a eukaryotic promoter involves a large number of factors that bind to a variety of cis-acting elements. Transcription factor: Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. Transcription factor: Any protein that is needed for the initiation of transcription, but which is not itself part of RNA polymerase, is defined as a transcription factor. 4. transcription factors

21 4.1 general or basal factors( 通用转录因子, 或基本 转录因子、普遍转录因子 ) 为所有 RNA 聚合酶Ⅱ转录起始所必需，在转录 起点周围和 RNA 聚合酶Ⅱ形成起始复合物，并协助 决定起始位点。 为所有 RNA 聚合酶Ⅱ转录起始所必需，在转录 起点周围和 RNA 聚合酶Ⅱ形成起始复合物，并协助 决定起始位点。 对于 RNA 聚合酶Ⅱ相应有 TF Ⅱ等 如 TF Ⅱ A 、 TF Ⅱ B 、 TF Ⅱ D 、 TF Ⅱ E 、 TF Ⅱ F. (RNA 聚合酶Ⅲ相 应有 TF Ⅲ ). 对于 RNA 聚合酶Ⅱ相应有 TF Ⅱ等 如 TF Ⅱ A 、 TF Ⅱ B 、 TF Ⅱ D 、 TF Ⅱ E 、 TF Ⅱ F. (RNA 聚合酶Ⅲ相 应有 TF Ⅲ ).

22 4.2 转录调节因子或上游因子 (upstream factor) 上游因子是 DNA 结合蛋白，广泛存在于细胞核中， 作用于含特异结合位点的启动子, 增加转录效率. 例如 : ① CAAT 区结合蛋白： CTF ( 即 NF-1) 最常见，与 CAAT 框相结合，促进 RNA 聚合酶Ⅱ指导的转录 起始（亦可促进腺病毒 DNA 复制）, 无组织特异性. ② GC 区结合蛋白 ① CAAT 区结合蛋白： CTF ( 即 NF-1) 最常见，与 CAAT 框相结合，促进 RNA 聚合酶Ⅱ指导的转录 起始（亦可促进腺病毒 DNA 复制）, 无组织特异性. ② GC 区结合蛋白 SP 1 识别 GGGCGG, 为 SV40 基因转录所必须。 SP 1 识别 GGGCGG, 为 SV40 基因转录所必须。

23 5. Process of Class II gene transcription Assembly of the initiation complex Assembly of the initiation complex Initiation Initiation Elongation Elongation Passing through the nucleosome Passing through the nucleosome Termination Termination Not clear. Sometimes, the termination sites are several kilobases away from the polyadenylation site. Not clear. Sometimes, the termination sites are several kilobases away from the polyadenylation site.

24 TF Ⅱ D 识别 TATA 。 TF Ⅱ D 是一个由 TBP （ TATA binding protein ）和 8 个以上 TBP 结合因子 组成的蛋白复合物。首先由 TF Ⅱ D 与 TATA 盒结合，再指导其它通用转录因子 装配在 DNA 分子的启动子上，形成转录 起始复合物。 [TF Ⅱ I 识别并结合 INR.]

25 Termination Eukaryotic transcription is similar to that of bacterial transcription in having the three phases: initiation, elongation and termination. Each of the three eukaryotic RNA polymerases recognizes different promoters and has a different mechanism of transcription termination.

26 3’3’5’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 6. Processing Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region 3’3’ AAAAA 3’ Poly A Tail 5’5’ G 5’ Cap Exon 2 Exon 3Exon 1 Int. 2Int. 1 m RNA processing achieves three things: m RNA processing achieves three things:  Addition of a 5 ’ cap-capping  Addition of a 3 ’ tail-tailing  Removal of introns-splicing

27 6.1 Eukaryotic Messenger RNA 5’ Capping

28 6.2 tailing

29 polyA 尾上游 10~35 个核苷酸处含有序列多聚腺苷 酸化信号 polyadenylation signal( often AAUAAA,especially in human,but less common in plants and fungi) 由一个酶识别该信号，然后在其下游方向切 割，接着由 poly (A) polymerase 加尾。 除该 AAUAAA 信号外，在被切除的部分（即切点下游 ）还有一个 GUGUGUG 的富含 G 、 U 区，也是一个多聚 腺苷酸化信号。 The poly A tail is found on most, but not all, eukaryotic RNA transcripts.

30 在细胞质内， mRNA 的 Poly(A) 尾巴与蛋白 质（ PABP ）结合，形成 mRNP 。 在细胞质内， mRNA 的 Poly(A) 尾巴与蛋白 质（ PABP ）结合，形成 mRNP 。 Function Function i. Stability: The poly(A) tail protects the mRNA molecule from degradation by exonucleases in the cytoplasm i. Stability: The poly(A) tail protects the mRNA molecule from degradation by exonucleases in the cytoplasm 高等真核生物多 A 尾长 200b ，低等真核约为 100b ，转移到 细胞质后，长度减少 30~50b 。组蛋白 mRNA 无多 A 尾，人工加 尾后，半衰期提高 10 倍。 高等真核生物多 A 尾长 200b ，低等真核约为 100b ，转移到 细胞质后，长度减少 30~50b 。组蛋白 mRNA 无多 A 尾，人工加 尾后，半衰期提高 10 倍。 ii.Aids in transcription termination, export of the mRNA from the nucleus. ii.Aids in transcription termination, export of the mRNA from the nucleus. iii. Aids in translation (enhancer of translation) iii. Aids in translation (enhancer of translation)

31 Role of the 3'-poly(A) sequence in translational regulation of mRNAs in Xenopus laevis oocytes Poly(A) 在实验中的应用：分离纯化 mRNA; cDNA 第一链合成；锚式 PCR 。

32 6.3 nuclear mRNA Splicing (see 8.1 )

33 other modifications methylation methylation RNA editing (see also gene regulation in eukaryotes) RNA editing (see also gene regulation in eukaryotes)

34 真核生物有 4 种 rRNA ，即 5.8S rRNA 、 18S rRNA 、 28S rRNA 和 5S rRNA 。其中，前三者的基因组成一个 转录单位，产生 47S 的前体，并很快转变成 45S 前体。真 核生物的 rRNA 的成熟过程比较缓慢，所以其加工的中 间体易于从各种细胞中分离得到。 真核生物有 4 种 rRNA ，即 5.8S rRNA 、 18S rRNA 、 28S rRNA 和 5S rRNA 。其中，前三者的基因组成一个 转录单位，产生 47S 的前体，并很快转变成 45S 前体。真 核生物的 rRNA 的成熟过程比较缓慢，所以其加工的中 间体易于从各种细胞中分离得到。 加工酶可能类似于原核的 RNAase Ⅲ。 加工酶可能类似于原核的 RNAase Ⅲ。 有关真核生物 tRNA 的切割处理了解还不多，可能 和原核生物有相似之处。 有关真核生物 tRNA 的切割处理了解还不多，可能 和原核生物有相似之处。 7.Processing Eukaryotic rRNA —— CLEAVAGE 7.Processing Eukaryotic rRNA —— 基因间序列的切 除（ RNA 的切割 CLEAVAGE ）

35 8. Splicing Splicing Removal of introns from the primary transcript and covalently joining of exons during the maturation of eukaryotic mRNA. Splicing Removal of introns from the primary transcript and covalently joining of exons during the maturation of eukaryotic mRNA.

36 RNA 的剪接 (splicing) 一个基因的外显子和内含子共同 转录在一条转录 产物中，然后将内含 子去除而把外显子连接起来形成成熟 的 RNA 分子，这一过程称为 RNA 剪接 （ RNA splicing ）。 一个基因的外显子和内含子共同 转录在一条转录 产物中，然后将内含 子去除而把外显子连接起来形成成熟 的 RNA 分子，这一过程称为 RNA 剪接 （ RNA splicing ）。 RNA splicing RNA splicing

37 8.1 nuclear mRNA Splicing -via Splicesome Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hnRNA Spliceosomes are structures that form within the nucleus to remove introns from eukaryotic hnRNA This structure is large, on the order of a ribosome subunit This structure is large, on the order of a ribosome subunit Like the ribosome, spliceosomes are composed of both protein and RNA Like the ribosome, spliceosomes are composed of both protein and RNA

38 Common Splicing Mechanism Exon 2Exon 1 Intron AGAGU 3’3’5’5’ 18-40 BP Branch site Left (donor) 5’ splice site Right (acceptor) 3’ splice site Py 80 NPy 80 Py 87 Pu 75 A Py 95 (Animal-Subscripts indicate percent frequency) U A C U A A C (Yeast) The branch sequence allows identification of the 3’ splice site

39 Common Splicing Mechanism 3’3’ Exon 2Exon 1 5’5’ A AG G U Intron lariat Following excision, the lariat is rapidly degraded

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41 8.2 Group I 8.2 Group I Intron self-splicing Splicing site U ↓ ………… G ↓ Splicing site U ↓ ………… G ↓ Introns have central core structure 如酵母细胞色素 b 基因 ScCOB GUU↓UAAUA………………UAAAUG↓CUC 如酵母细胞色素 b 基因 ScCOB GUU↓UAAUA………………UAAAUG↓CUC Group I catalytic introns are large self-splicing ribozymes. They catalyze their own excision from mRNA, tRNA and rRNA precursors in a wide range of organisms. Group I catalytic introns are large self-splicing ribozymes. They catalyze their own excision from mRNA, tRNA and rRNA precursors in a wide range of organisms.

42 Distribution of Group I introns is weirdly broad but irregular Found in: 1. Mitochondria and plastid genomes of plants and protists (rRNA, tRNA and mRNA genes) 2. Nucleus of certain protists, fungi and lichens (rRNA genes) 3. Eubacteria (tRNA genes) & phages 4. Metazoans - only in mitochondrial genes of a few anthozoans (e.g., sea anemone)

43 反应条件： 反应条件： 一价阳离子、二价阳离子 一价阳离子、二价阳离子 GTP （或 GDP 、 GMP 或鸟嘌呤核 苷），不能由其它的核苷酸代替， 实验中用放射性同位素标记跟踪 G 的去向。 剪接机制 : 鸟苷添加转酯反应 剪接机制 : 鸟苷添加转酯反应

44 two successive transesterification steps The 3'-OH of a guanosine (G, GMP, GDP, GTP all function) acts as a nucleophile, attacks the phosphate at the 5' exon-intron junction, and covalently binds to the excised intron. This step requires metal ions for folding and catalysis. The 3'-OH of a guanosine (G, GMP, GDP, GTP all function) acts as a nucleophile, attacks the phosphate at the 5' exon-intron junction, and covalently binds to the excised intron. This step requires metal ions for folding and catalysis. 3‘-OH of the released 5' -exon becomes a nucleophile and attacks the 3' junction phosphate, completing the splice. the second transesterification results in the joining of the two exons. 3‘-OH of the released 5' -exon becomes a nucleophile and attacks the 3' junction phosphate, completing the splice. the second transesterification results in the joining of the two exons.

45 8.3 Group II intron self-splicing ↓ GUGYG ………… YnAY ↓ ↓ GUGYG ………… YnAY ↓ （ Y =pyrimidine ） （ Y =pyrimidine ） 第二类内含子最早发现于线粒体，它的剪接位 点与核基因 mRNA 的内含子类似，近似遵循 GT-AG 规则。 (evolutionary precursors to nuclear mRNA/spliceosomal introns; OR : It is thought that pre-mRNA splicing may have evolved from group II introns. ) 第二类内含子最早发现于线粒体，它的剪接位 点与核基因 mRNA 的内含子类似，近似遵循 GT-AG 规则。 (evolutionary precursors to nuclear mRNA/spliceosomal introns; OR : It is thought that pre-mRNA splicing may have evolved from group II introns. )

46 Distribution Group II intron is a class of intron found in rRNA, tRNA, mRNA of organelles in fungi, plants, protists, and mRNA in bacteria. Group II intron is a class of intron found in rRNA, tRNA, mRNA of organelles in fungi, plants, protists, and mRNA in bacteria.

47 Self-splicing occurs in vitro (for a few of the introns studied to date), but protein machinery is probably required in vivo. 剪接机制 套索（ lariat ）形成转酯反应 套索（ lariat ）形成转酯反应 不需 -OH 的鸟核苷（酸），不需一价阳离子，仅需 5mM 左右的 Mg2+ 和少量亚精胺，不需额外能量输入。 不需 -OH 的鸟核苷（酸），不需一价阳离子，仅需 5mM 左右的 Mg2+ 和少量亚精胺，不需额外能量输入。

48 Distribution of Group II introns is a little more restrictive Mitochondrial and plastid genomes of plants and protists (rRNA, tRNA and mRNA genes) EubacteriaArchae Not found in nuclear or viral genes

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50 酵母细胞核中 400 个 tRNA 基因中约有 40 个是断裂基因。这些基因 均只有一个内含子，位于与反密码子的 3 ‘ 侧相隔一个核苷酸之处， 长度为 14 至 46bp 。不同氨基酸的 tRNA 基因中的内含子不相同，因 此，剪接酶类看来并不能识别任何共同顺序。剪接酶系识别的是 tRNA 前体的二级结构。 剪接酶与原核 tRNA 后加工过程中的酶有相 似之外。 8.4 Yeast tRNA enzymatic splicing Another rare form of splicing. The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together. Ribonucleasesligases

51 剪接主要分两步进行。第一步先由内切 酶切断磷酸二酯键，该反应属非典型的核 酸酶反应，不需要 ATP ；第二步由 RNA 连 接酶将断端连接形成熟的 tRNA 分子，该反 应需要 ATP 的参与。 剪接主要分两步进行。第一步先由内切 酶切断磷酸二酯键，该反应属非典型的核 酸酶反应，不需要 ATP ；第二步由 RNA 连 接酶将断端连接形成熟的 tRNA 分子，该反 应需要 ATP 的参与。

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53 Exercises 1. Answer these questions about eukaryotic RNA. a. What is a cap? a. What is a cap? b. At which end of the mRNA is the poly(A)? b. At which end of the mRNA is the poly(A)? c. Are there eukaryotic mRNA molecules that c. Are there eukaryotic mRNA molecules that do not contain either feature? do not contain either feature? 2. a. What are intervening sequences or introns? b. What is meant by mRNA "splicing"? b. What is meant by mRNA "splicing"? 3. a. What two regions are common to most prokaryotic promoters? prokaryotic promoters? b. What consensus sequence is present in a b. What consensus sequence is present in a large number of eukaryotic promoters? large number of eukaryotic promoters?

54 4. Match the following with their role in transcription in eukaryotic cells. 4. Match the following with their role in transcription in eukaryotic cells. _____ The RNA synthesized after RNA polymerase copies both the exons and the interons of a gene. the interons of a gene. _____ The RNA produced after noncoding regions (interons) are excised and coding regions (exons) are joined together by complexes of coding regions (exons) are joined together by complexes of ribonucleoproteins called spliceosomes. ribonucleoproteins called spliceosomes. _____ An unusual nucleotide, 7-methylguanylate, that is added to the 5' end of the pre-mRNA early in transcription. It helps ribosomes attach for the pre-mRNA early in transcription. It helps ribosomes attach for translation. translation. _____ Non-protein coding regions of DNA that are not part of the code for the final protein that are interspersed among the coding regions of the final protein that are interspersed among the coding regions of DNA in most genes of higher eukaryotic cells. DNA in most genes of higher eukaryotic cells. _____ The coding regions of DNA in most genes of higher eukaryotic cells that actually code for the final protein. that actually code for the final protein. _____ A series of 100-250 adenine ribonucleotides that is added to the 3' end of the pre-mRNA. This series of nucleotides is thought to help end of the pre-mRNA. This series of nucleotides is thought to help transport the mRNA out of the nucleus and may stabilize the mRNA transport the mRNA out of the nucleus and may stabilize the mRNA against degradation in the cytoplasm. against degradation in the cytoplasm. a. introns b. exons c. precurser mRNA d. cap e. poly-A tail f. mature mRNA a. introns b. exons c. precurser mRNA d. cap e. poly-A tail f. mature mRNA